Prostate-specific membrane antigen binding proteins and related compositions and methods

ABSTRACT

The present invention relates to mono-specific and multi-specific polypeptide therapeutics that specifically target cells expressing prostate-specific membrane antigen (PSMA) and are useful for the treatment of prostate cancer (e.g., castrate-resistant prostate cancer), tumor-related angiogenesis or benign prostatic hyperplasia (BPH). In one embodiment, the multi-specific polypeptide therapeutics bind both PSMA-expressing cells and the T-cell receptor complex on T cells to induce target-dependent T-cell cytotoxicity, activation and proliferation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/113,353, filed Feb. 12, 2014, which is the National Stage ofInternational Application No. PCT/US2012/034575, filed Apr. 20, 2012,which claims priority to U.S. Provisional Patent Application No.61/478,449, filed Apr. 22, 2011, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to mono-specific and multi-specificprotein therapeutics that specifically target cells expressingprostate-specific membrane antigen (PSMA) and are useful for thetreatment of disorders characterized by overexpression of PSMA, such as,for example, prostate cancer (e.g., castrate-resistant prostate cancer),tumor-related angiogenesis, or benign prostatic hyperplasia (BPH). Inone embodiment, the multi-specific protein therapeutic binds bothPSMA-expressing cells and the T-cell receptor complex on T cells toinduce target-dependent T-cell cytotoxicity, activation andproliferation.

ACCOMPANYING SEQUENCE LISTING

The contents of the text file (Name: “APVO_028_02US_SeqList.txt”; Size:269,000 bytes; Date of Creation: May 3, 2017) submitted electronicallyherewith are incorporated herein by reference in their entirety.

BACKGROUND

Prostate-specific Membrane Antigen (PSMA), also known as glutamatecarboxypeptidase II and N-acetylated alpha-linked acidic dipeptidase 1,is a dimeric type II transmembrane glycoprotein belonging to the M28peptidase family encoded by the gene FOLH1 (folate hydrolase 1). Theprotein acts as a glutamate carboxypeptidase on different alternativesubstrates, including the nutrient folate and the neuropeptideN-acetyl-1-aspartyl-1-glutamate and is expressed in a number of tissuessuch as the prostate, and to a lesser extent, the small intestine,central and peripheral nervous system and kidney. The gene encoding PSMAis alternatively spliced to produce at least three variants. A mutationin this gene may be associated with impaired intestinal absorption ofdietary folates, resulting in low blood folate levels and consequenthyperhomocysteinemia. Expression of this protein in the brain may beinvolved in a number of pathological conditions associated withglutamate excitotoxicity.

PSMA is a well-established, highly restricted prostate-cancer-relatedcell membrane antigen. In prostate cancer cells, PSMA is expressed1000-fold higher than on normal prostate epithelium (Su et al., CancerRes. 1995 44:1441-1443). Expression of PSMA increases with prostatecancer progression and is highest in metastatic disease, hormonerefractory cases, and higher-grade lesions (Israeli et al., Cancer Res.1994, 54:1807-1811; Wright et al., Urologic Oncology: Seminars andOriginal Investigations 1995 1:18-28; Wright et al., Urology 199648:326-332; Sweat et al., Urology 1998 52:637-640). Additionally, PSMAis abundantly expressed on the neovasculature of a variety of othersolid tumors, including bladder, pancreas, melanoma, lung and kidneycancers, but not on normal neovasculature (Chang et al., Urology 200157:801-805; Divgi et al., Clin. Cancer Res. 1998 4:2729-3279).

PSMA has been shown to be an important target for immunologicalapproaches such as vaccines or directed therapy with monoclonalantibodies. Unlike other prostate-restricted molecules that aresecretory proteins (PSA, prostatic acid phosphatase), PSMA is anintegral cell-surface membrane protein that is not secreted, which makesit an ideal target for antibody therapy. PROSTASCINT® (capromabpendetide) is an ¹¹¹In-labelled anti-PSMA murine monoclonal antibodyapproved by the FDA for imaging and staging of newly diagnosed andrecurrent prostate cancer patients (Hinkle et al., Cancer 1998,83:739-747). However, capromab binds to an intracellular epitope ofPSMA, requiring internalization or exposure of the internal domain ofPSMA, therefore preferentially binding apoptotic or necrosing cells(Troyer et al., Urologic Oncology: Seminars and Original Investigations1995 1:29-37; Troyer et al., Prostate 1997 30:232-242). As a result,capromab may not be of therapeutic benefit (Liu et al., Cancer Res. 199757:3629-3634).

Other monoclonal antibodies which target the external domain of PSMAhave been developed (e.g., J591, J415, J533, and E99) (Liu et al.,Cancer Res. 1997 57:3629-3634). Radiolabelled J591 is currently inclinical trials (Tagawa et al., Cancer 2010 116(54):1075). However,evidence suggests that PSMA may act as a receptor mediating theinternalization of a putative ligand. PSMA undergoes internalizationconstitutively, and PSMA-specific antibodies can induce and/or increasethe rate of internalization, which then causes the antibodies toaccumulate in the endosomes (Liu et al., Cancer Res. 1998 58:4055-4060).While PSMA-specific internalizing antibodies may aid in the developmentof therapeutics to target the delivery of toxins, drugs, orradioisotopes to the interior of prostate cancer cells (Tagawa et al.,Cancer 2010 116(54):1075), PSMA-specific antibodies utilizing native orengineered effector mechanisms (e.g., antibody-dependent cell-mediatedcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC),antibody-dependent cell-mediated phagocytosis (ADCP), or re-directedT-cell cytotoxicity (RTCC)) are problematic since the PSMA-specificantibody may be internalized before it is recognized by effector cells.

SUMMARY OF THE INVENTION

In one embodiment, the present disclosure provides a prostate-specificmembrane antigen (PSMA)-binding polypeptide comprising, in order fromamino-terminus to carboxyl-terminus, (a) a PSMA-binding domain thatspecifically binds human PSMA, (b) a hinge region, and (c) animmunoglobulin constant region. In certain embodiments, suitablePSMA-binding domains include binding domains that compete for binding tohuman PSMA with a single chain Fv (scFv) having the amino acid sequenceset forth in SEQ ID NO:21. In certain embodiments, the PSMA-bindingpolypeptide is capable of forming a dimer with a second, identicalpolypeptide chain through association between the respectiveimmunoglobulin constant regions and/or hinge regions.

In certain embodiments, the PSMA-binding domain comprises (i) animmunoglobulin light chain variable region comprising CDRs LCDR1, LCDR2,and LCDR3, and/or (ii) an immunoglobulin heavy chain variable regioncomprising CDRs HCDR1, HCDR2, and HCDR3. In certain variations, LCDR3has the amino acid sequence set forth in SEQ ID NO:17 and/or HCDR3 hasthe amino acid sequence set forth in SEQ ID NO:11; in some suchembodiments, LCDR1 and LCDR2 have the amino acid sequences as set forthin SEQ ID NO:15 and SEQ ID NO:16, respectively, and/or HCDR1 and HCDR2have the amino acid sequences as set forth in SEQ ID NO:9 and SEQ IDNO:10, respectively. In another variation, (i) the light chain variableregion comprises an amino acid sequence that is at least 90%, at least95%, at least 99%, or 100% identical to the amino acid sequence setforth in SEQ ID NO:5 or SEQ ID NO:23; and/or (ii) the heavy chainvariable region comprises an amino acid sequence that is at least 90%,at least 95%, at least 99%, or 100% identical to the amino acid sequenceset forth in SEQ ID NO:2, SEQ ID NO:25, or SEQ ID NO:27. One or both ofthe light and heavy chain variable regions can be humanized.

In certain variations, the PSMA-binding domain is a single chain Fv(scFv) comprising the immunoglobulin light and heavy chain variableregions disclosed herein. In certain embodiments, PSMA-binding scFvsinclude, for example, scFvs comprising an amino acid sequence that is atleast 90%, at least 95%, at least 99%, or 100% identical to the aminoacid sequence set forth in SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:30, SEQID NO:31, SEQ ID NO:34, or SEQ ID NO:35. In certain embodiments, theheavy chain variable region of the scFv is carboxyl-terminal to thelight chain variable region (also referred to herein as a “VL-VHorientation”). In some embodiments of an scFv having a VL-VHorientation, the scFv comprises an amino acid sequence that is at least90%, at least 95%, at least 99%, or 100% identical to the amino acidsequence set forth in SEQ ID NO:21, SEQ ID NO:30, or SEQ ID NO:31. Thelight chain variable region and heavy chain variable region of the scFvcan be joined by a peptide linker such as, for example, a peptide linkercomprising an amino acid sequence (Gly₄Ser)_(n), wherein n=1-5 (SEQ IDNO:165).

In some embodiments of a PSMA-binding polypeptide disclosed herein, thehinge region is derived from an immunoglobulin hinge region, such as,for example, an immunoglobulin hinge region of IgG1, IgG2, IgG3, IgG4,IgA1, IgA2, or IgD. Such an immunoglobulin hinge region can be either awild-type or an altered immunoglobulin hinge region.

In further embodiments of a PSMA-binding polypeptide disclosed herein,the immunoglobulin constant region comprises immunoglobulin CH2 and CH3domains, such as, for example, immunoglobulin CH2 and CH3 domains ofIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In another embodiment, theimmunoglobulin constant region comprises immunoglobulin CH2 and CH3domains and the constant region does not comprise an immunoglobulin CH1domain.

In certain variations, a PSMA-binding polypeptide disclosed hereinincludes at least one effector function selected from antibody-dependentcell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC). In some embodiments, the hinge region is derived from animmunoglobulin hinge region and the immunoglobulin constant regioncomprises immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, orIgG4. In another embodiment, the immunoglobulin hinge region is derivedfrom the hinge region of IgG1 and the immunoglobulin constant regioncomprises immunoglobulin CH2 and CH3 domains of IgG1.

In some embodiments, a PSMA-binding polypeptide disclosed hereincomprises an amino acid sequence that is at least 90%, at least 95%, atleast 99%, or 100% identical to the amino acid sequence set forth in SEQID NO:38, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:70, or SEQID NO:72.

In still further embodiments, a PSMA-binding polypeptide disclosedherein further includes (d) a second hinge region carboxyl-terminal tothe immunoglobulin constant region, and (e) a second binding domaincarboxyl-terminal to the second hinge region. In some embodiments,second hinge regions include those derived from a stalk region of a typeII C lectin or an immunoglobulin hinge region. In certain variations,the second hinge region has an amino acid sequence as set forth in SEQID NO:63, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66.

In another embodiment, the present disclosure provides aprostate-specific membrane antigen (PSMA)-binding polypeptide thatspecifically binds human PSMA and comprises a first binding domaincomprising (i) an immunoglobulin light chain variable region comprisingCDRs LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chainvariable region comprising CDRs HCDR1, HCDR2, and HCDR3; wherein LCDR3has the amino acid sequence set forth in SEQ ID NO:17 and/or HCDR3 hasthe amino acid sequence set forth in SEQ ID NO:11. In some embodiments,LCDR1 and LCDR2 have the amino acid sequences as set forth in SEQ IDNO:15 and SEQ ID NO:16, respectively, and/or HCDR1 and HCDR2 have theamino acid sequences as set forth in SEQ ID NO:9 and SEQ ID NO:10,respectively. In some variations, LCDR1, LCDR2, and LCDR3 have the aminoacid sequences as set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ IDNO:17, respectively; and HCDR1, HCDR2, and HCDR3 have the amino acidsequences as set forth in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11,respectively. In some variations, (i) the light chain variable regioncomprises an amino acid sequence that is at least 90%, at least 95%, atleast 99%, or 100% identical to the amino acid sequence set forth in SEQID NO:5 or SEQ ID NO:23; and/or (ii) the heavy chain variable regioncomprises an amino acid sequence that is at least 90%, at least 95%, atleast 99%, or 100% identical to the amino acid sequence set forth in SEQID NO:2, SEQ ID NO:25, or SEQ ID NO:27. In certain embodiments, thelight chain variable region is encoded by a nucleic acid sequence thatis at least 90%, at least 95%, at least 99%, or 100% identical to thenucleic acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, or SEQ IDNO:22; and/or the heavy chain variable region is encoded by a nucleicacid sequence that is at least 90%, at least 95%, at least 99%, or 100%identical to the nucleic acid sequence set forth in SEQ ID NO:1, SEQ IDNO:24, or SEQ ID NO:26. One or both of the light and heavy chainvariable regions can be humanized. In some embodiments, the PSMA-bindingpolypeptide is capable of forming a dimer with a second, identicalpolypeptide chain.

In certain embodiments disclosed herein, the first binding domain is asingle chain Fv (scFv) comprising the immunoglobulin light and heavychain variable regions. In some embodiments, PSMA-binding scFvs include,for example, scFvs comprising an amino acid sequence that is at least90%, at least 95% at least 99%, or 100% identical to the amino acid setforth in SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:34, or SEQ ID NO:35. In certain embodiments, the heavy chain variableregion of the scFv is carboxyl-terminal to the light chain variableregion (a “VL-VH orientation”). In some embodiments of an scFv having aVL-VH orientation, the scFv comprises an amino acid sequence that is atleast 90%, at least 95%, at least 99%, or 100% identical to the aminoacid sequence set forth in SEQ ID NO:21, SEQ ID NO:30, or SEQ ID NO:31.The light chain variable region and heavy chain variable region of thescFv can be joined by a peptide linker such as, for example, a peptidelinker comprising an amino acid sequence (Gly₄Ser)_(fl), wherein n=1-5(SEQ ID NO:165).

In certain embodiments, the PSMA-binding polypeptide further includes animmunoglobulin constant region. For example, in some variations, theimmunoglobulin constant region comprises immunoglobulin CH2 and CH3domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In somevariations, the PSMA-binding polypeptide further includes one or morehinge regions. In certain embodiments, the hinge region can be derived,for instance, from a stalk region of a type II C lectin or from animmunoglobulin hinge region.

In another embodiment, the PSMA binding polypeptide comprises, in orderfrom amino to carboxyl-terminus, a first binding domain, a hinge region,and an immunoglobulin constant region. A PSMA-binding polypeptide inthis format can also be referred to as a PSMA-specific SMIP molecule.General SMIP configurations are provided, for example, in US PatentApplication Publication Nos. 2003/0133939, 2003/0118592, and2005/0136049, which are incorporated herein in their entirety byreference.

In another embodiment, the orientation of the polypeptide is reversedsuch that the polypeptide comprises, in order from amino tocarboxyl-terminus, an immunoglobulin constant region, a hinge region anda first binding domain. In this orientation, the polypeptide can also bereferred to as a PSMA-specific PIMS molecule. General PIMSconfigurations are provided, for example, in US Patent ApplicationPublication No. 2009/0148447, which is incorporated herein in itsentirety by reference. In some embodiments, a PSMA-binding polypeptidehaving an immunoglobulin constant region and, optionally, a hinge regionas disclosed herein is capable of forming a dimer with a second,identical polypeptide chain through association between the respectiveimmunoglobulin constant regions and/or hinge regions.

In another embodiment, the PSMA-binding polypeptide includes a secondbinding domain, such as, e.g., a single-chain Fv (scFv). For example, insome variations, the PSMA-binding polypeptide comprises, in order fromamino-terminus to carboxyl-terminus or in order from carboxyl-terminusto amino-terminus, (a) a first binding domain, (b) a first hinge region,(c) an immunoglobulin constant region, (d) a second hinge region, and(e) a second binding domain.

In yet another embodiment, the present disclosure provides aPSMA-binding polypeptide as in other embodiments disclosed herein andcomprising an additional binding domain, e.g., a second binding domain,wherein the second binding domain specifically binds a T cell. Incertain embodiments, the second binding domain specifically binds a Tcell receptor (TCR) complex or a component thereof. In some embodiments,the second binding domain includes those that specifically bind CD3,e.g., CD3ε. In certain variations, the second binding domain competesfor binding to CD3 with the CRIS-7 or HuM291 monoclonal antibody. Insome such variations, the second binding domain comprises animmunoglobulin light chain variable region and an immunoglobulin heavychain variable region derived from the CRIS-7 or HuM291 monoclonalantibody. For example, in certain embodiments, the light and heavy chainvariable regions of the second binding domain are humanized variableregions comprising, respectively, the light and heavy chain CDRs of theCRIS-7 or HuM291 monoclonal antibody. In another embodiment, the lightand heavy chain variable regions of the second binding domain areselected from (a) a light chain variable region comprising an amino acidsequence that is at least 90%, at least 95%, at least 99%, or 100%identical to the amino acid sequence set forth in residues 139-245 ofSEQ ID NO:47 and a heavy chain variable region comprising an amino acidsequence that is at least 90%, at least 95%, at least 99%, or 100%identical to the amino acid sequence set forth in residues 1-121 of SEQID NO:47; and (b) a light chain variable region comprising an amino acidsequence that is at least 90%, at least 95%, at least 99%, or 100%identical to the amino acid sequence set forth in residues 634-740 ofSEQ ID NO:78 and a heavy chain variable region comprising an amino acidsequence that is at least 90%, at least 95%, at least 99%, or 100%identical to the amino acid sequence set forth in residues 496-616 ofSEQ ID NO:78.

In certain embodiments of a PSMA-binding polypeptide comprising a secondbinding domain, the second binding domain is a single-chain Fv (scFv).For example, in some embodiments of a second binding domain comprisinglight and heavy chain variable regions derived from the CRIS-7monoclonal antibody, the second binding domain is a scFv comprising anamino acid sequence that is at least 90%, at least 95%, at least 99%, or100% identical to an amino acid sequence selected from (i) the aminoacid sequence set forth in residues 1-245 of SEQ ID NO:47, and (ii) theamino acid sequence set forth in residues 496-742 of SEQ ID NO:78. Insome such embodiments, the PSMA-binding polypeptide comprises an aminoacid sequence that is at least 90%, at least 95%, at least 99%, or 100%identical to the amino acid sequence set forth in SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ IDNO:82, SEQ ID NO:84, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, or SEQID NO:164.

In another embodiment, the present disclosure provides a dimericPSMA-binding protein comprising first and second polypeptide chains,wherein each of said polypeptide chains is a PSMA-binding polypeptide asin any of the embodiments disclosed herein.

In another embodiment, the present disclosure provides a PSMA-bindingpolypeptide comprising, in order from amino-terminus tocarboxyl-terminus, (a) a binding domain that specifically binds humanPSMA, (b) a hinge region, (c) an immunoglobulin constant region, and (d)an immunoglobulin heterodimerization domain. The heterodimerizationdomain can comprise, for example, an immunoglobulin CH1 domain or animmunoglobulin CL domain. In certain embodiments, the PSMA-bindingdomain competes for binding to human PSMA with a single chain Fv (scFv)having the amino acid sequence set forth in SEQ ID NO:21. In certainembodiments, the PSMA-binding domains include, e.g., the PSMA-bindingdomains disclosed above.

In some embodiments, the hinge region is derived from an immunoglobulinhinge region, such as, for example, an immunoglobulin hinge region ofIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. Such an immunoglobulin hingeregion can be either a wild-type or an altered immunoglobulin hingeregion. In further embodiments, the immunoglobulin constant regioncomprises immunoglobulin CH2 and CH3 domains, such as, for example,immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1,IgA2, IgD, or any combination thereof; an immunoglobulin CH3 domain ofIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM or any combinationthereof; or immunoglobulin CH3 and CH4 domains of IgE, IgM or acombination thereof.

In certain embodiments, a PSMA-binding polypeptide includes at least oneeffector function selected from antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In somesuch embodiments, the hinge region is derived from an immunoglobulinhinge region and the immunoglobulin constant region comprisesimmunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, or IgG4. In morespecific variations, the immunoglobulin hinge region is derived from thehinge region of IgG1 and the immunoglobulin constant region comprisesimmunoglobulin CH2 and CH3 domains of IgG1.

In certain embodiments, a PSMA-binding polypeptide comprises an aminoacid sequence that is at least 90%, at least 95%, at least 99%, or 100%%identical to the amino acid sequence set forth in SEQ ID NO:46, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, or SEQ ID NO:61.

In another embodiment, the present disclosure provides a PSMA-bindingprotein comprising two, non-identical polypeptide chains that associateby way of heterodimerization domains (e.g., immunoglobulinheterodimerization domains) to form a heterodimer. In some embodiments,the heterodimeric PSMA binding protein comprises a first polypeptidechain comprising, in order from amino-terminus to carboxyl-terminus, (a)a first binding domain that specifically binds PSMA, (b) a first hingeregion, (c) a first immunoglobulin constant region, and (d) a firstimmunoglobulin heterodimerization domain; and a second single chainpolypeptide comprising, in order from amino-terminus tocarboxyl-terminus, (a) a second hinge region, (b) a secondimmunoglobulin sub-region, and (c) a second immunoglobulinheterodimerization domain that is different from the firstimmunoglobulin heterodimerization domain of the first polypeptide chain,wherein the first and second immunoglobulin heterodimerization domainsassociate with each other to form a heterodimer. In certain embodiments,the PSMA-binding domain competes for binding to human PSMA with a singlechain Fv (scFv) having the amino acid sequence set forth in SEQ IDNO:21. In certain embodiments, the PSMA-binding domains include, e.g.,the PSMA-binding domains disclosed above.

In certain embodiments, heterodimerization domains include domainscomprising either an immunoglobulin CH1 domain or an immunoglobulin CLdomain. In some such embodiments, the first immunoglobulinheterodimerization domain comprises a first immunoglobulin CH1 domainand the second immunoglobulin heterodimerization domain comprises afirst immunoglobulin CL domain. Alternatively, in other embodiments, thefirst immunoglobulin heterodimerization domain comprises a firstimmunoglobulin CL domain and the second immunoglobulinheterodimerization domain comprises a first immunoglobulin CH1 domain.

In some embodiments, at least one of the first and second hinge regionsis derived from an immunoglobulin hinge region, such as, for example, animmunoglobulin hinge region of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, orIgD. Such an immunoglobulin hinge region can be either a wild-type or analtered immunoglobulin hinge region. In further embodiments, at leastone of the first and second immunoglobulin constant regions comprisesimmunoglobulin CH2 and CH3 domains, such as, for example, immunoglobulinCH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, or anycombination thereof; an immunoglobulin CH3 domain of IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, IgD, IgE, IgM or any combination thereof; orimmunoglobulin CH3 and CH4 domains of IgE, IgM or a combination thereof.

In certain variations of a heterodimeric PSMA-binding protein asdisclosed herein, one or both of the first and second polypeptide chainsinclude at least one effector function selected from antibody-dependentcell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC). In some such embodiments, each of the first and second hingeregions is derived from an immunoglobulin hinge region and each of thefirst and second immunoglobulin constant regions comprisesimmunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, or IgG4. Incertain embodiments, each of the first and second hinge regions isderived from the hinge region of IgG1 and each of the first and secondimmunoglobulin constant region comprises immunoglobulin CH2 and CH3domains of IgG1.

In some embodiments of a heterodimeric PSMA-binding protein as disclosedherein, the second polypeptide chain further includes a second bindingdomain. For example, the second polypeptide chain can further comprise asecond binding domain amino-terminal to the second hinge region.

In certain variations, a heterodimeric PSMA-binding protein as disclosedherein can be monospecific monospecific for PSMA). Alternatively, inother embodiments, the heterodimeric PSMA-binding protein ismultispecific. For instance, each polypeptide chain of the heterodimercan comprise different binding domains, e.g., the first polypeptidechain comprising the PSMA-binding domain and the second polypeptidechain comprising a second binding (e.g., amino-terminal to the secondhinge region) that is specific for a second target antigen that isdifferent from PSMA.

In some embodiments of a multispecific, heterodimeric PSMA-bindingprotein, the second binding domain specifically binds a T-cell. Incertain embodiments, T-cell-binding domains include, e.g., theadditional binding domains and second binding domains disclosed above.In certain embodiments of a heterodimeric PSMA-binding proteincomprising a second binding domain that specifically binds a T-cell, forexample, (a) the first polypeptide chain comprises an amino acidsequence that is at least 90%, at least 95%, at least 99%, or 100%identical to the amino acid sequence set forth in SEQ ID NO: 46 and thesecond polypeptide chain comprises an amino acid sequence that is atleast 90%, at least 95%, at least 99%, or 100% identical to the aminoacid sequence set forth in SEQ ID NO: 47; (b) the first polypeptidechain comprises an amino acid sequence that is at least 90%, at least95%, at least 99%, or 100% identical to the amino acid sequence setforth in SEQ ID NO: 58 and the second polypeptide chain comprises anamino acid sequence that is at least 90%, at least 95%, at least 99%, or100% identical to the amino acid sequence set forth in SEQ ID NO: 57;(c) the first polypeptide chain comprises an amino acid sequence that isat least 90%, at least 95%, at least 99%, or 100% identical to the aminoacid sequence set forth in SEQ ID NO: 59 and the second polypeptidechain comprises an amino acid sequence that is at least 90%, at least95%, at least 99%, or 100% identical to the amino acid sequence setforth in SEQ ID NO: 57; (d) the first polypeptide chain comprises anamino acid sequence that is at least 99%, at least 95%, at least 99%, or100% identical to the amino acid sequence set forth in SEQ ID NO: 60 andthe second polypeptide chain comprises an amino acid sequence that is atleast 90%, at least 95%, at least 99%, or 100% identical to the aminoacid sequence set forth in SEQ ID NO: 47; or (e) the first polypeptidechain comprises an amino acid sequence that is at least 90%, at least95%, at least 99%, or 100% identical to the amino acid sequence setforth in SEQ ID NO: 61 and the second polypeptide chain comprises anamino acid sequence that is at least 90%, at least 95%, at least 99%, or100% identical to the amino acid sequence set forth in SEQ ID NO: 47.

In certain embodiments of a dimeric or heterodimeric PSMA-bindingprotein as disclosed herein, the PSMA-binding protein exhibits increasedserum half-life, reduced internalization by a cell expressing PSMA,and/or increased time of persistence on the surface of the cellexpressing PSMA as compared to the murine monoclonal antibody 107-1A4.

In another embodiment, the present disclosure provides an isolatednucleic acid encoding a PSMA-binding polypeptide. For example, incertain variations, the nucleic acid comprises the nucleotide sequenceset forth in SEQ ID NO NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24,SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:44,SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ ID: NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75,SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:157,SEQ ID NO:159, SEQ ID NO:161, or SEQ ID NO:163.

In another embodiment, the present disclosure provides an expressionvector for expressing a PSMA-binding polypeptide or protein as disclosedherein in a recombinant host cell. In some embodiments, the expressionvector comprises a nucleic acid segment encoding the PSMA-bindingpolypeptide, wherein the nucleic acid segment is operably linked toregulatory sequences suitable for expression of the nucleic acid segmentin a host cell. In some embodiments, the nucleic acid segment comprisesthe nucleotide sequence set forth in SEQ ID NO NO:18, SEQ ID NO:20, SEQID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ ID:NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, or SEQ ID NO:163. Inother embodiments, the expression vector comprises first and secondexpression units, wherein the first and second expression unitsrespectively comprise first and second nucleic acid segments encodingthe first and second polypeptide chains of a heterodimeric PSMA-bindingprotein as in certain embodiments disclosed herein, and wherein thefirst and second nucleic acid segments are operably linked to regulatorysequences suitable for expression of the nucleic acid segments in a hostcell. In certain variations, (a) the first nucleic acid segmentcomprises the nucleotide sequence set forth in SEQ ID NO:44 and thesecond nucleic acid segment comprises the nucleotide sequence set forthin SEQ ID NO:45; (b) the first nucleic acid segment comprises thenucleotide sequence set forth in SEQ ID NO:53 and the second nucleicacid segment comprises the nucleotide sequence set forth in SEQ IDNO:52; (c) the first nucleic acid segment comprises the nucleotidesequence set forth in SEQ ID NO:54 and the second nucleic acid segmentcomprises the nucleotide sequence set forth in SEQ ID NO:52; (d) thefirst nucleic acid segment comprises the nucleotide sequence set forthin SEQ ID NO:55 and the second nucleic acid segment comprises thenucleotide sequence set forth in SEQ ID NO:45; or (e) the first nucleicacid segment comprises the nucleotide sequence set forth in SEQ ID NO:56and the second nucleic acid segment comprises the nucleotide sequenceset forth in SEQ ID NO:45.

In another embodiment, the present disclosure provides a recombinanthost cell comprising an expression vector disclosed herein.

In another embodiment, the present disclosure provides a method forproducing a PSMA-binding polypeptide or protein. For example, in someembodiments, the method is for producing a PSMA-binding polypeptide asdisclosed herein. In certain embodiments, the method generally includesculturing a recombinant host cell comprising an expression vector,wherein the expression vector comprises a nucleic acid segment thatencodes the PSMA-binding polypeptide and is operably linked toregulatory sequences suitable for expression of the nucleic acid segmentin the host cell, and wherein the culturing is under conditions wherebythe nucleic acid segment is expressed, thereby producing thePSMA-binding polypeptide. In certain variations, the nucleic acidsegment comprises the nucleotide sequence set forth in SEQ ID NO NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:50,SEQ ID NO:53, SEQ ID: NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:69,SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79,SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161,or SEQ ID NO:163. In certain embodiments, the method further includesrecovering the PSMA-binding polypeptide.

In some embodiments, the method is for producing a dimeric PSMA-bindingprotein as disclosed herein. In certain variations, the nucleic acidsegment of the expression vector encodes the PSMA-binding polypeptide asdisclosed herein, and the culturing is under conditions whereby thenucleic acid segment is expressed and the encoded PSMA-bindingpolypeptide is produced as a dimeric PSMA-binding protein. The methodcan further include recovering the dimeric PSMA-binding protein.

In other embodiments, the method is for producing a heterodimericPSMA-binding protein disclosed herein. In certain embodiments, themethod generally includes culturing a recombinant host cell comprisingfirst and second expression units, wherein the first and secondexpression units respectively comprise first and second nucleic acidsegments encoding the first and second polypeptide chains of aheterodimeric PSMA-binding protein as set forth herein, wherein thefirst and second nucleic acid segments are operably linked to regulatorysequences suitable for expression of the nucleic acid segments in a hostcell, and wherein the culturing is under conditions whereby the firstand second nucleic acid segments are expressed and the encodedpolypeptide chains are produced as the heterodimeric PSMA-bindingprotein. In some embodiments, the method further includes recovering theheterodimeric PSMA-binding protein.

In another embodiment, the present disclosure provides a compositioncomprising any of the PSMA-binding polypeptides or proteins as set forthherein and a pharmaceutically acceptable carrier, diluent, or excipient.

In another embodiment, the present disclosure provides a method forinducing antibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC) against a cell expressing PSMA.For example, in some embodiments, a method for inducing ADCC or CDCagainst the cell expressing PSMA includes contacting the PSMA-expressingcell with a dimeric PSMA-binding protein comprising first and secondpolypeptide chains, wherein each of the polypeptide chains is aPSMA-binding polypeptide as disclosed herein, and wherein the contactingis under conditions whereby ADCC or CDC against the PSMA-expressing cellis induced. In other embodiments, a method for inducing ADCC or CDCagainst the PSMA-expressing cell includes contacting the cell with aheterodimeric PSMA-binding protein as in paragraph [0031], wherein thecontacting is under conditions whereby ADCC or CDC against thePSMA-expressing cell is induced.

In another embodiment, the present disclosure provides a method forinducing redirected T-cell cytotoxicity (RTCC) against a cell expressingPSMA. In some variations, a method for inducing RTCC against the cellexpressing PSMA includes contacting the PSMA-expressing cell with adimeric PSMA-binding protein comprising first and second polypeptidechains, wherein each of said polypeptide chains is a PSMA-bindingpolypeptide disclosed herein, and wherein the contacting is underconditions whereby RTCC against the PSMA-expressing cell is induced. Inother embodiments, a method for inducing RTCC against thePSMA-expressing cell includes contacting the cell with a heterodimericPSMA-binding protein as disclosed herein, wherein the contacting isunder conditions whereby RTCC against the PSMA-expressing cell isinduced.

In another embodiment, the present disclosure provides a method fortreating a disorder in a subject, wherein the disorder is characterizedby overexpression of PSMA. In some embodiments, the method includesadministering to the subject a therapeutically effective amount of adimeric PSMA-binding protein disclosed above. In some such embodiments,the first and second polypeptide chains of the dimeric PSMA-bindingprotein is a PSMA-binding polypeptide, e.g., as disclosed above, and thedimeric PSMA-binding protein induces redirected T-cell cytotoxicity(RTCC) in the subject. In other variations, the method includesadministering to the subject a therapeutically effective amount of aheterodimeric PSMA-binding protein, e.g., as disclosed above. In somevariations, the heterodimeric PSMA-binding protein is a protein asdisclosed above, and the heterodimeric PSMA-binding protein induces RTCCin the subject. In certain embodiments of the disclosed methods, thedisorder is a cancer such as, for example, prostate cancer (e.g.,castrate-resistant prostate cancer), colorectal cancer, gastric cancer,clear cell renal carcinoma, bladder cancer, or lung cancer. In someembodiments, the disorder is a prostate disorder such as, e.g., prostatecancer or benign prostatic hyperplasia. In other variations, thedisorder is an neovascular disorder. The neovascular disorder to betreated can be, for example, a cancer characterized by solid tumorgrowth such as, e.g., clear cell renal carcinoma, colorectal cancer,bladder cancer, and lung cancer.

These and other embodiments and/or other aspects of the invention willbecome evident upon reference to the following detailed description ofthe invention and the attached drawings.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph illustrating the results of a binding study used tocompare the parent 107-1A4 murine antibody (TSC045) with TSC085, TSC092and TSC122 in PSMA(+) (LNCaP) and PSMA(−) (DU-145) prostate cancer celllines.

FIG. 2A is a graph illustrating the results of a binding study used tocompare humanized TSC188 and TSC189 in PSMA(+) (C4-2) and PSMA(−)(DU-145) prostate cancer cell lines.

FIG. 2B is a graph illustrating the results of a binding study used tocompare binding of humanized SCORPION molecules TSC194 and TSC199 tothat of parent humanized SMIP molecules TSC188 and TSC189 and chimericInterceptor molecule TSC122 in PSMA(+) (C4-2) and PSMA(−) (DU-145)prostate cancer cell lines.

FIG. 3 is a graph illustrating the results of internalizationexperiments comparing the parent 107-1A4 murine antibody to PSMA-bindingproteins built on Interceptor and SMIP scaffolds.

FIG. 4 is a graph illustrating potent target-dependent cytotoxicactivity over 24 hours observed with the chimeric TSC122 Interceptormolecule at decreasing concentrations (300, 100, 30, 10 and 0 pM) in thepresence of T cells from human blood from two different donors (labeledas AG and VV).

FIG. 5 is a graph illustrating cytotoxicity activity of TSC200, TSC202,TSC204 alongside the parent chimeric Interceptor molecule TSC122.

FIG. 6 is a graph illustrating T-cell cytotoxicity mediated by humanized107-1A4 SCORPION molecules (TSC194, TSC199, TSC212, TSC213) compared tothe chimeric Interceptor molecule TSC122.

FIGS. 7A and 7B are graphs illustrating target-dependent proliferationof CD4+ T-cells (FIG. 7A) and CD8+ T-cells (FIG. 7B) induced byanti-PSMA bispecific molecules (TSC194, TSC199, TSC202 and TSC122)reacting with C4-2 cells.

FIGS. 8A-8C are graphs illustrating competitive binding studies of mAbsJ591 and J415 versus 107-1A4 mAb and chimeric and humanized 107-1A4 SMIPmolecules to PSMA on C4-2 cells. Specifically, FIG. 8A shows the resultsof a competitive binding assay to determine if the humanized J591antibody (Hu591) competes with the binding of 107-1A4, J591 or J415murine antibodies to PSMA on C4-2 cells; FIG. 8B shows the results of acompetitive binding assay to determine if the three murine antibodiescompete with the binding of the chimeric 107-1A4 SMIP molecule (TSC085)to PSMA on C4-2 cells; and FIG. 8C shows the results of a competitivebinding assay to determine if the three murine antibodies compete withthe binding of the humanized 107-1A4 SMIP molecule (TSC189) to PSMA onC4-2 cells.

DETAILED DESCRIPTION OF THE INVENTION I. General Description

The invention provides PSMA-binding polypeptides and proteins thatspecifically bind prostate-specific membrane antigen (PSMA).Administration of a therapeutically effective amount of a PSMA-bindingpolypeptide or protein of the invention to a patient in need thereof isuseful for treatment of certain disorders associated with theover-expression of PSMA, including certain cancers and prostatedisorders. In one embodiment, the PSMA-binding polypeptide or proteinsimultaneously bind a target cell over-expressing PSMA and a T-cell,thereby “cross-linking” the target cell over-expressing PSMA and theT-cell. The binding of both domains to their targets elicits potenttarget-dependent redirected T-cell cytotoxicity (RTCC) (e.g., inducestarget-dependent T-cell cytotoxicity, T-cell activation and T-cellproliferation).

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited herein, including but notlimited to patents, patent applications, articles, books, and treatises,are hereby expressly incorporated by reference in their entirety for anypurpose. In the event that one or more of the incorporated documents orportions of documents define a term that contradicts that term'sdefinition in the application, the definition that appears in thisapplication controls.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. As used herein, “about” means±20% of the indicatedrange, value, or structure, unless otherwise indicated. It should beunderstood that the terms “a” and “an” as used herein refer to “one ormore” of the enumerated components unless otherwise indicated. The useof the alternative (e.g., “or”) should be understood to mean either one,both, or any combination thereof of the alternatives. As used herein,the terms “include” and “comprise” are used synonymously. In addition,it should be understood that the polypeptides comprising the variouscombinations of the components (e.g., domains or regions) andsubstituents described herein, are disclosed by the present applicationto the same extent as if each polypeptide was set forth individually.Thus, selection of particular components of individual polypeptides iswithin the scope of the present disclosure.

II. Definitions

As used herein, the term “binding domain” or “binding region” refers tothe domain, region, portion, or site of a protein, polypeptide,oligopeptide, or peptide that possesses the ability to specificallyrecognize and bind to a target molecule, such as an antigen, ligand,receptor, substrate, or inhibitor (e.g., CD3, PSMA). Exemplary bindingdomains include single-chain antibody variable regions (e.g., domainantibodies, sFv, scFv, scFab), receptor ectodomains, and ligands (e.g.,cytokines, chemokines). In certain embodiments, the binding domaincomprises or consists of an antigen binding site (e.g., comprising avariable heavy chain sequence and variable light chain sequence or threelight chain complementary determining regions (CDRs) and three heavychain CDRs from an antibody placed into alternative framework regions(FRs) (e.g., human FRs optionally comprising one or more amino acidsubstitutions). A variety of assays are known for identifying bindingdomains of the present disclosure that specifically bind a particulartarget, including Western blot, ELISA, phage display library screening,and BIACORE® interaction analysis. As used herein, a PSMA-bindingpolypeptide can have a “first binding domain” and, optionally, a “secondbinding domain.” In certain embodiments, the “first binding domain” is aPSMA-binding domain and, depending on the particular polypeptide format(e.g., SMIP or PIMS), can be located at either the amino- orcarboxyl-terminus. In certain embodiments comprising both the first andsecond binding domains, the second binding domain is a T cell bindingdomain such as a scFv derived from a mouse monoclonal antibody (e.g.,CRIS-7) that binds to a T cell surface antigen (e.g., CD3). In otherembodiments, the second binding domain is a second PSMA-binding domain.In yet other embodiments, the second binding domain is a binding domainother than a PSMA-binding domain or a T cell binding domain.

A binding domain “specifically binds” a target if it binds the targetwith an affinity or K_(a) (i.e., an equilibrium association constant ofa particular binding interaction with units of 1/M) equal to or greaterthan 10⁵ M⁻¹, while not significantly binding other components presentin a test sample. Binding domains can be classified as “high affinity”binding domains and “low affinity” binding domains. “High affinity”binding domains refer to those binding domains with a K_(a) of at least10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least10¹¹ M⁻¹, at least 10¹² M⁻¹, or at least 10¹³ M⁻¹. “Low affinity”binding domains refer to those binding domains with a K_(a) of up to 10⁷M⁻¹, up to 10⁶ M⁻¹, up to 10⁵ M⁻¹. Alternatively, affinity can bedefined as an equilibrium dissociation constant (K_(d)) of a particularbinding interaction with units of M (e.g., 10⁻⁵ M to 10⁻¹³ M).Affinities of binding domain polypeptides and single chain polypeptidesaccording to the present disclosure can be readily determined usingconventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y.Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, or theequivalent).

“CD3” is known in the art as a multi-protein complex of six chains (see,e.g., Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999),which are subunits of the T cell receptor complex. In mammals, the CD3subunits of the T cell receptor complex are a CD3γ chain, a CD3δ chain,two CD3ε chains, and a homodimer of CD3ζ chains. The CD3γ, CD3δ, andCD3ε chains are highly related cell surface proteins of theimmunoglobulin superfamily containing a single immunoglobulin domain.The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains arenegatively charged, which is a characteristic that allows these chainsto associate with the positively charged T cell receptor chains. Theintracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain asingle conserved motif known as an immunoreceptor tyrosine-basedactivation motif or ITAM, whereas each CD3ζ chain has three. It isbelieved the ITAMs are important for the signaling capacity of a TCRcomplex. CD3 as used in the present disclosure can be from variousanimal species, including human, monkey, mouse, rat, or other mammals.

As used herein, a “conservative substitution” is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are well-knownin the art (see, e.g., WO 97/09433, page 10, published Mar. 13, 1997;Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY(1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and CellPress, Cambridge, Mass. (1990), p. 8). In certain embodiments, aconservative substitution includes a leucine to serine substitution.

As used herein, the term “derivative” refers to a modification of one ormore amino acid residues of a peptide by chemical or biological means,either with or without an enzyme, e.g., by glycosylation, alkylation,acylation, ester formation, or amide formation.

As used herein, a polypeptide or amino acid sequence “derived from” adesignated polypeptide or protein refers to the origin of thepolypeptide. In certain embodiments, the polypeptide or amino acidsequence which is derived from a particular sequence (sometimes referredto as the “starting” or “parent” or “parental” sequence) has an aminoacid sequence that is essentially identical to the starting sequence ora portion thereof, wherein the portion consists of at least 10-20 aminoacids, at least 20-30 amino acids, or at least 30-50 amino acids, or atleast 50-150 amino acids, or which is otherwise identifiable to one ofordinary skill in the art as having its origin in the starting sequence.

Polypeptides derived from another polypeptide can have one or moremutations relative to the starting polypeptide, e.g., one or more aminoacid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions. The polypeptide can comprise an amino acid sequence which isnot naturally occurring. Such variations necessarily have less than 100%sequence identity or similarity with the starting polypeptide. In oneembodiment, the variant will have an amino acid sequence from about 60%to less than 100% amino acid sequence identity or similarity with theamino acid sequence of the starting polypeptide. In another embodiment,the variant will have an amino acid sequence from about 75% to less than100%, from about 80% to less than 100%, from about 85% to less than100%, from about 90% to less than 100%, from about 95% to less than 100%amino acid sequence identity or similarity with the amino acid sequenceof the starting polypeptide.

As used herein, unless otherwise provided, a position of an amino acidresidue in a variable region of an immunoglobulin molecule is numberedaccording to the Kabat numbering convention (Kabat, Sequences ofProteins of Immunological Interest, 5^(th) ed. Bethesda, Md.: PublicHealth Service, National Institutes of Health (1991)), and a position ofan amino acid residue in a constant region of an immunoglobulin moleculeis numbered according to EU nomenclature (Ward et al., 1995 Therap.Immunol. 2:77-94).

As used herein, the term “dimer” refers to a biological entity thatconsists of two subunits associated with each other via one or moreforms of intramolecular forces, including covalent bonds (e.g.,disulfide bonds) and other interactions (e.g., electrostaticinteractions, salt bridges, hydrogen bonding, and hydrophobicinteractions), and is stable under appropriate conditions (e.g., underphysiological conditions, in an aqueous solution suitable forexpressing, purifying, and/or storing recombinant proteins, or underconditions for non-denaturing and/or non-reducing electrophoresis). A“heterodimer” or “heterodimeric protein,” as used herein, refers to adimer formed from two different polypeptides. A heterodimer does notinclude an antibody formed from four polypeptides (i.e., two lightchains and two heavy chains). A “homodimer” or “homodimeric protein,” asused herein, refers to a dimer formed from two identical polypeptides.

As used herein, a “hinge region” or a “hinge” refers to a polypeptidederived from (a) an interdomain region of a transmembrane protein (e.g.,a type I transmembrane protein); or (b) a stalk region of a type IIC-lectin. For example, a hinge region can be derived from an interdomainregion of an immunoglobulin superfamily member; suitable hinge regionswithin this particular class include (i) immunoglobulin hinge regions(made up of, for example, upper and/or core region(s)) or functionalvariants thereof, including wild-type and altered immunoglobulin hinges,and (ii) regions (or functional variants thereof) that connectimmunoglobulin V-like or immunoglobulin C-like domains.

A “wild-type immunoglobulin hinge region” refers to a naturallyoccurring upper and middle hinge amino acid sequences interposed betweenand connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) orinterposed between and connecting the CH1 and CH3 domains (for IgE andIgM) found in the heavy chain of an antibody. In certain embodiments, awild type immunoglobulin hinge region sequence is human, and cancomprise a human IgG hinge region.

An “altered wild-type immunoglobulin hinge region” or “alteredimmunoglobulin hinge region” refers to (a) a wild type immunoglobulinhinge region with up to 30% amino acid changes (e.g., up to 25%, 20%,15%, 10%, or 5% amino acid substitutions or deletions), or (b) a portionof a wild type immunoglobulin hinge region that has a length of about 5amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 amino acids) up to about 120 amino acids (for instance,having a length of about 10 to about 40 amino acids or about 15 to about30 amino acids or about 15 to about 20 amino acids or about 20 to about25 amino acids), has up to about 30% amino acid changes (e.g., up toabout 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% amino acid substitutionsor deletions or a combination thereof), and has an IgG core hinge regionas disclosed in PCT/US2010/62436 and PCT/US2010/62404.

As used herein, the term “humanized” refers to a process of making anantibody or immunoglobulin binding proteins and polypeptides derivedfrom a non-human species (e.g., mouse or rat) less immunogenic tohumans, while still retaining antigen-binding properties of the originalantibody, using genetic engineering techniques. In some embodiments, thebinding domain(s) of an antibody or immunoglobulin binding proteins andpolypeptides (e.g., light and heavy chain variable regions, Fab, scFv)are humanized. Non-human binding domains can be humanized usingtechniques known as CDR grafting (Jones et al., Nature 321:522 (1986))and variants thereof, including “reshaping” (Verhoeyen, et al., 1988Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337;Tempest, et al., Bio/Technol 1991 9:266-271), “hyperchimerization”(Queen, et al., 1989 Proc Nati Acad Sci USA 86:10029-10033; Co, et al.,1991 Proc Nati Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol148:1149-1154), and “veneering” (Mark, et al., “Derivation oftherapeutically active humanized and veneered anti-CD18 antibodies. In:Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition totherapeutic potential. New York: Plenum Press, 1994: 291-312). Ifderived from a non-human source, other regions of the antibody orimmunoglobulin binding proteins and polypeptides, such as the hingeregion and constant region domains, can also be humanized.

An “immunoglobulin dimerization domain” or “immunoglobulinheterodimerization domain”, as used herein, refers to an immunoglobulindomain of a polypeptide chain that preferentially interacts orassociates with a different immunoglobulin domain of a secondpolypeptide chain, wherein the interaction of the differentimmunoglobulin heterodimerization domains substantially contributes toor efficiently promotes heterodimerization of the first and secondpolypeptide chains (i.e., the formation of a dimer between two differentpolypeptide chains, which is also referred to as a “heterodimer”). Theinteractions between immunoglobulin heterodimerization domains“substantially contributes to or efficiently promotes” theheterodimerization of first and second polypeptide chains if there is astatistically significant reduction in the dimerization between thefirst and second polypeptide chains in the absence of the immunoglobulinheterodimerization domain of the first polypeptide chain and/or theimmunoglobulin heterodimerization domain of the second polypeptidechain. In certain embodiments, when the first and second polypeptidechains are co-expressed, at least 60%, at least about 60% to about 70%,at least about 70% to about 80%, at least 80% to about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second polypetpidechains form heterodimers with each other. Representative immunoglobulinheterodimerization domains include an immunoglobulin CH1 domain, animmunoglobulin CL domain (e.g., Cκ or Cλ isotypes), or derivativesthereof, including wild type immunoglobulin CH1 and CL domains andaltered (or mutated) immunoglobulin CH1 and CL domains, as providedtherein.

An “immunoglobulin constant region” or “constant region” is a termdefined herein to refer to a peptide or polypeptide sequence thatcorresponds to or is derived from part or all of one or more constantregion domains. In certain embodiments, the immunoglobulin constantregion corresponds to or is derived from part or all of one or moreconstant region domains, but not all constant region domains of a sourceantibody. In certain embodiments, the constant region comprises IgG CH2and CH3 domains, e.g., IgG1 CH2 and CH3 domains. In certain embodiments,the constant region does not comprise a CH1 domain. In certainembodiments, the constant region domains making up the constant regionare human. In some embodiments (for example, in certain variations of aPSMA-binding polypeptide or protein comprising a second binding domainthat specifically binds CD3 or another T cell surface antigen), theconstant region domains of a fusion protein of this disclosure lack orhave minimal effector functions of antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement activation and complement-dependentcytotoxicity (CDC), while retaining the ability to bind some F_(c)receptors (such as F_(c)Rn, the neonatal Fc receptor) and retaining arelatively long half life in vivo. In other variations, a fusion proteinof this disclosure includes constant domains that retain such effectorfunction of one or both of ADCC and CDC. In certain embodiments, abinding domain of this disclosure is fused to a human IgG1 constantregion, wherein the IgG1 constant region has one or more of thefollowing amino acids mutated: leucine at position 234 (L234), leucineat position 235 (L235), glycine at position 237 (G237), glutamate atposition 318 (E318), lysine at position 320 (K320), lysine at position322 (K322), or any combination thereof (numbering according to EU). Forexample, any one or more of these amino acids can be changed to alanine.In a further embodiment, an IgG1 Fc domain has each of L234, L235, G237,E318, K320, and K322 (according to EU numbering) mutated to an alanineL234A, L235A, G237A, E318A, K320A, and K322A, respectively), andoptionally an N297A mutation as well (i.e., essentially eliminatingglycosylation of the CH2 domain).

“Fc region” or “Fc domain” refers to a polypeptide sequencecorresponding to or derived from the portion of a source antibody thatis responsible for binding to antibody receptors on cells and the C1qcomponent of complement. Fc stands for “fragment crystalline,” thefragment of an antibody that will readily form a protein crystal.Distinct protein fragments, which were originally described byproteolytic digestion, can define the overall general structure of animmunoglobulin protein. As originally defined in the literature, the Fcfragment consists of the disulfide-linked heavy chain hinge regions,CH2, and CH3 domains. However, more recently the term has been appliedto a single chain consisting of CH3, CH2, and at least a portion of thehinge sufficient to form a disulfide-linked dimer with a second suchchain. For a review of immunoglobulin structure and function, seePutnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp.49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, theterm Fc includes variants of naturally occuring sequences.

As used here the term “SMIP” is used to refer to protein scaffold asgenerally disclosed in, for example, in US Patent ApplicationPublication Nos. 2003/0133939, 2003/0118592, and 2005/0136049, which areincorporated herein by reference in their entirety. The “PSMA-specificSMIP molecules” or “SMIP molecules” described in the Examples andthroughout the disclosure herein should be understood to be PSMA-bindingproteins comprising SMIP scaffolding, e.g., in order from amino tocarboxyl-terminus, a first binding domain, a hinge region, and animmunoglobulin constant constant region.

As used here the term “PIMS” is used to refer to protein scaffold asgenerally disclosed in, for example, in US Patent ApplicationPublication No. 2009/0148447, which is incorporated herein in itsentirety by reference. The “PSMA-specific PIMS molecules” or “PIMSmolecules” described in the Examples and throughout the disclosureherein should be understood to be PSMA-binding proteins comprising PIMSscaffolding, e.g., in order from amino to carboxyl-terminus, animmunoglobulin constant region, a hinge region and a first bindingdomain.

As used herein, the term “Interceptor” is used to refer to amonospecific or multispecific heterodimeric protein scaffold asgenerally disclosed in PCT applications PCT/US2010/62436 andPCT/US2010/62404, which are incorporated herein in their entirety. The“PSMA-specific Interceptor molecules” or “Interceptor molecules”described in the Examples and throughout the disclosure herein should beunderstood to be PSMA-binding proteins comprising Interceptorscaffolding, e.g., two non-identical polypeptide chains, eachpolypeptide chain comprising an immunoglobulin heterodimerizationdomain. The interfacing immunoglobulin heterodimerization domains aredifferent. In one embodiment, the immunoglobulin heterodimerizationdomain comprises a CH1 domain or a derivative thereof. In anotherembodiment, the immunoglobulin heterodimerization domain comprises a CLdomain or a derivative thereof. In one embodiment, the CL domain is a Cκor Cλ isotype or a derivative thereof.

As used herein, “SCORPION”, is a term used to refer to a multi-specificbinding protein scaffold. SCORPION™ is a trademark of Emergent ProductDevelopment Seattle, LLC. Multi-specific binding proteins andpolypeptides are disclosed, for instance, in PCT Application PublicationNo. WO 2007/146968, U.S. Patent Application Publication No.2006/0051844, PCT Application Publication No. WO 2010/040105, PCTApplication Publication No. WO 2010/003108, and U.S. Pat. No. 7,166,707,which are incorporated herein by reference in their entirety. A SCORPIONpolypeptide comprises two binding domains (the domains can be designedto specifically bind the same or different targets), two hinge regions,and an immunoglobulin constant region. SCORPION proteins are homodimericproteins comprising two identical, disulfide-bonded SCORPIONpolypeptides. The “PSMA-specific SCORPION molecules” or “SCORPIONmolecules” described in the Examples and throughout the disclosureherein should be understood to be PSMA-binding proteins comprisingSCORPION scaffolding, e.g., two binding domains (the domains can bedesigned to specifically bind the same or different targets), two hingeregions, and an immunoglobulin constant region.

As used herein, the “stalk region” of a type II C-lectin refers to theportion of the extracellular domain of the type II C-lectin that islocated between the C-type lectin-like domain (CTLD; e.g., similar toCTLD of natural killer cell receptors) and the transmembrane domain. Forexample, in the human CD94 molecule (GenBank™ Accession No. AAC50291.1,PRI Nov. 30, 1995), the extracellular domain corresponds to amino acidresidues 34-179, whereas the CTLD corresponds to amino acid residues61-176. Accordingly, the stalk region of the human CD94 moleculeincludes amino acid residues 34-60, which is found between the membraneand the CTLD (see Boyington et al., Immunity 10:75, 1999; fordescriptions of other stalk regions, see also Beavil et al., Proc.Nat'l. Acad. Sci. USA 89:753, 1992; and Figdor et al., Nature Rev.Immunol. 2:77, 2002). These type II C-lectins can also have from six to10 junction amino acids between the stalk region and the transmembraneregion or the CTLD. In another example, the 233 amino acid human NKG2Aprotein (GenBank™ Accession No. P26715.1, PRI Jun. 15, 2010) has atransmembrane domain ranging from amino acids 71-93 and an extracellulardomain ranging from amino acids 94-233. The CTLD is comprised of aminoacids 119-231, and the stalk region comprises amino acids 99-116, whichis flanked by junctions of five and two amino acids. Other type IIC-lectins, as well as their extracellular ligand-bind domains,interdomain or stalk regions, and CTLDs are known in the art (see, e.g.,GenBank™ Accession Nos. NP_001993.2; AAH07037.1, PRI Jul. 15, 2006;NP_001773.1, PRI Jun. 20, 1010; AAL65234.1, PRI Jan. 17, 2002, andCAA04925.1, PRI Nov. 14, 2006, for the sequences of human CD23, CD69,CD72, NKG2A and NKG2D and their descriptions, respectively).

As used herein, the “interdomain region” of a transmembrane protein(e.g., a type I transmembrane protein) refers to a portion of theextracellular domain of the transmembrane protein that is locatedbetween two adjacent domains. Examples of interdomain regions includeregions linking adjacent Ig domains of immunoglobulin superfamilymembers (e.g., an immunoglobulin hinge region from IgG, IgA, IgD, orIgE; the region linking the IgV and IgC2 domains of CD2; or the regionlinking the IgV and IgC domains of CD80 or CD86). Another example of aninterdomain region is the region linking the non-Ig and IgC2 domain ofCD22, a type I sialic acid-binding Ig-like lectin.

A polypeptide region “derived from” a stalk region of a type IIC-lectin, or “derived from” a transmembrane protein interdomain region(e.g., an immunoglobulin hinge region), refers to an about five to about150 amino acid sequence, an about 5 to about 100 amino acid sequence, anabout 5 to about 50 amino acid sequence, an about 5 to about 40 aminoacid sequence, an about 5 to about 30 amino acid sequence, an about 5 toabout 25 amino acid sequence, an about 5 to about 20 amino acidsequence, an about 10 to about 25 amino acid sequence, an about 10 toabout 20 amino acid sequence, about 8 to about 20 amino acid sequence,about 9 to about 20 amino acid sequence, about 10 to about 20 amino acidsequence, about 11 to about 20 amino acid sequence, about 12 to about 20amino acid sequence, about 13 to about 20 amino acid sequence, about 14to about 20 amino acid sequence, about 15 to about 20 amino acidsequence, or an about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acid sequence, wherein all or at least a portion ofwhich includes (i) a wild-type stalk region or interdomain regionsequence; (ii) a fragment of the wild-type stalk region or interdomainregion sequence; (iii) a polypeptide having at least 80%, 85%, 90%, or95% amino acid sequence identity with either (i) or (ii); or (iv) either(i) or (ii) in which one, two, three, four, or five amino acids have adeletion, insertion, substitution, or any combination thereof, forinstance, the one or more changes are substitutions or the one or moremutations include only one deletion. In some embodiments, a derivativeof a stalk region is more resistant to proteolytic cleavage as comparedto the wild-type stalk region sequence, such as those derived from abouteight to about 20 amino acids of NKG2A, NKG2D, CD23, CD64, CD72, orCD94.

As used herein, the term “junction amino acids” or “junction amino acidresidues” refers to one or more (e.g., about 2-10) amino acid residuesbetween two adjacent regions or domains of a polypeptide, such asbetween a hinge and an adjacent immunoglobulin constant region orbetween a hinge and an adjacent binding domain or between a peptidelinker that links two immunoglobulin variable domains and an adjacentimmunoglobulin variable domain. Junction amino acids can result from theconstruct design of a polypeptide (e.g., amino acid residues resultingfrom the use of a restriction enzyme site during the construction of anucleic acid molecule encoding a polypeptide).

As used herein, the phrase a “linker between CH3 and CH1 or CL” refersto one or more (e.g., about 2-12, about 2-10, about 4-10, about 5-10,about 6-10, about 7-10, about 8-10, about 9-10, about 8-12, about 9-12,or about 10-12) amino acid residues between the C-terminus of a CH3domain (e.g., a wild type CH3 or a mutated CH3) and the N-terminus of aCH1 domain or CL domain (e.g., Ck).

As used herein, the term “patient in need” refers to a patient at riskof, or suffering from, a disease, disorder or condition that is amenableto treatment or amelioration with a PSMA-binding protein or polypeptideor a composition thereof provided herein.

As used herein, the term “peptide linker” refers to an amino acidsequence that connects a heavy chain variable region to a light chainvariable region and provides a spacer function compatible withinteraction of the two sub-binding domains so that the resultingpolypeptide retains a specific binding affinity to the same targetmolecule as an antibody that comprises the same light and heavy chainvariable regions. In certain embodiments, a linker is comprised of fiveto about 35 amino acids, for instance, about 15 to about 25 amino acids.

As used herein, the term “pharmaceutically acceptable” refers tomolecular entities and compositions that do not produce allergic orother serious adverse reactions when administered using routes wellknown in the art. Molecular entities and compositions approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans are considered to be“pharmaceutically acceptable.”

As used herein, the term “promoter” refers to a region of DNA involvedin binding RNA polymerase to initiate transcription.

As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or“polynucleotide” refer to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the terms encompass nucleic acids containinganalogues of natural nucleotides that have similar binding properties asthe reference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081;Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al.(1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The termnucleic acid is used interchangeably with gene, cDNA, and mRNA encodedby a gene. As used herein, the terms “nucleic acid,” “nucleic acidmolecule,” or “polynucleotide” are intended to include DNA molecules(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of theDNA or RNA generated using nucleotide analogs, and derivatives,fragments and homologs thereof.

The term “expression” refers to the biosynthesis of a product encoded bya nucleic acid. For example, in the case of nucleic acid segmentencoding a polypeptide of interest, expression involves transcription ofthe nucleic acid segment into mRNA and the translation of mRNA into oneor more polypeptides.

The terms “expression unit” and “expression cassette” are usedinterchangeably herein and denote a nucleic acid segment encoding apolypeptide of interest and capable of providing expression of thenucleic acid segment in a host cell. An expression unit typicallycomprises a transcription promoter, an open reading frame encoding thepolypeptide of interest, and a transcription terminator, all in operableconfiguration. In addition to a transcriptional promoter and terminator,an expression unit can further include other nucleic acid segments suchas, e.g., an enhancer or a polyadenylation signal.

The term “expression vector,” as used herein, refers to a nucleic acidmolecule, linear or circular, comprising one or more expression units.In addition to one or more expression units, an expression vector canalso include additional nucleic acid segments such as, for example, oneor more origins of replication or one or more selectable markers.Expression vectors are generally derived from plasmid or viral DNA, orcan contain elements of both.

As used herein, the term “sequence identity” refers to a relationshipbetween two or more polynucleotide sequences or between two or morepolypeptide sequences. When a position in one sequence is occupied bythe same nucleic acid base or amino acid residue in the correspondingposition of the comparator sequence, the sequences are said to be“identical” at that position. The percentage “sequence identity” iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of “identical” positions. The number of “identical”positions is then divided by the total number of positions in thecomparison window and multiplied by 100 to yield the percentage of“sequence identity.” Percentage of “sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window. Thecomparison window for nucleic acid sequences can be, for instance, atleast 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or morenucleic acids in length. The comparison windon for polypeptide sequencescan be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids inlength. In order to optimally align sequences for comparison, theportion of a polynucleotide or polypeptide sequence in the comparisonwindow can comprise additions or deletions termed gaps while thereference sequence is kept constant. An optimal alignment is thatalignment which, even with gaps, produces the greatest possible numberof “identical” positions between the reference and comparator sequences.Percentage “sequence identity” between two sequences can be determinedusing the version of the program “BLAST 2 Sequences” which was availablefrom the National Center for Biotechnology Information as of Sep. 1,2004, which program incorporates the programs BLASTN (for nucleotidesequence comparison) and BLASTP (for polypeptide sequence comparison),which programs are based on the algorithm of Karlin and Altschul (Proc.Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing “BLAST 2Sequences,” parameters that were default parameters as of Sep. 1, 2004,can be used for word size (3), open gap penalty (11), extension gappenalty (1), gap dropoff (50), expect value (10) and any other requiredparameter including but not limited to matrix option. Two nucleotide oramino acid sequences are considered to have “substantially similarsequence identity” or “substantial sequence identity” if the twosequences have at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity relative to each other.

As used herein, a “polypeptide” or “polypeptide chain” is a single,linear and contiguous arrangement of covalently linked amino acids. Itdoes not include two polypeptide chains that link together in anon-linear fashion, such as via an interchain disulfide bond (e.g., ahalf immunoglobulin molecule in which a light chain links with a heavychain via a disulfide bond). Polypeptides can have or form one or moreintrachain disulfide bonds. With regard to polypeptides as describedherein, reference to amino acid residues corresponding to thosespecified by SEQ ID NO includes post-translational modifications of suchresidues.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein can also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentscan be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

As used herein, “small modular immunopharmaceutical proteins” or SMIPrefers to a protein scaffold generally disclosed, for instance, U.S.Patent Publication Nos. 2003/0133939, 2003/0118592, and 2005/0136049.SMIP™ is a trademark of Emergent Product Development Seattle LLC. A SMIPprotein can comprise a polypeptide chain having a binding domain, ahinge region and an immunoglobulin constant region.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl-terminus of thereference sequence, but is not necessarily at the carboxyl-terminus ofthe complete polypeptide.

“T cell receptor” (TCR) is a molecule found on the surface of T cellsthat, along with CD3, is generally responsible for recognizing antigensbound to major histocompatibility complex (MHC) molecules. It consistsof a disulfide-linked heterodimer of the highly variable α and β chainsin most T cells. In other T cells, an alternative receptor made up ofvariable γ and δ chains is expressed. Each chain of the TCR is a memberof the immunoglobulin superfamily and possesses one N-terminalimmunoglobulin variable domain, one immunoglobulin constant domain, atransmembrane region, and a short cytoplasmic tail at the C-terminal end(see Abbas and Lichtman, Cellular and Molecular Immunology (5th Ed.),Editor: Saunders, Philadelphia, 2003; Janeway et al., Immunobiology: TheImmune System in Health and Disease, 4^(th) Ed., Current BiologyPublications, p 148, 149, and 172, 1999). TCR as used in the presentdisclosure can be from various animal species, including human, mouse,rat, or other mammals.

“TCR complex,” as used herein, refers to a complex formed by theassociation of CD3 chains with other TCR chains. For example, a TCRcomplex can be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains,a homodimer of CD3ζ chains, a TCRα chain, and a TCRβ chain.Alternatively, a TCR complex can be composed of a CD3γ chain, a CD3δchain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRγ chain, and aTCRβ chain.

“A component of a TCR complex,” as used herein, refers to a TCR chain(i.e., TCRα, TCRβ, TCRγ or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε orCD3ζ), or a complex formed by two or more TCR chains or CD3 chains(e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complexof CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex ofTCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC,” as usedherein, refer to a cell-mediated process in which nonspecific cytotoxiccells that express FcγRs (e.g., monocytic cells such as Natural Killer(NK) cells and macrophages) recognize bound antibody (or other proteincapable of binding FcγRs) on a target cell and subsequently cause lysisof the target cell. In principle, any effector cell with an activatingFcγR can be triggered to mediate ADCC. The primary cells for mediatingADCC are NK cells, which express only FcγRIII, whereas monocytes,depending on their state of activation, localization, ordifferentiation, can express FcγRI, FcγRII, and FcγRIII. For a review ofFcγR expression on hematopoietic cells, see, e.g., Ravetch et al., 1991,Annu. Rev. Immunol., 9:457-92.

The term “having ADCC activity,” as used herein in reference to apolypeptide or protein, means that the polypeptide or protein (forexample, one comprising an immunoglobulin hinge region and animmunoglobulin constant region having CH2 and CH3 domains, such asderived from IgG (e.g., IgG1)), is capable of mediatingantibody-dependent cell-mediated cytotoxicity (ADCC) through binding ofa cytolytic Fc receptor (e.g., FcγRIII) on a cytolytic immune effectorcell expressing the Fc receptor (e.g., an NK cell).

“Complement-dependent cytotoxicity” and “CDC,” as used herein, refer toa process in which components in normal serum (“complement”), togetherwith an antibody or other C1q-complement-binding protein bound to atarget antigen, exhibit lysis of a target cell expressing the targetantigen. Complement consists of a group of serum proteins that act inconcert and in an orderly sequence to exert their effect.

The terms “classical complement pathway” and “classical complementsystem,” as used herein, are synonymous and refer to a particularpathway for the activation of complement. The classical pathway requiresantigen-antibody complexes for initiation and involves the activation,in an orderly fashion, of nine major protein components designated C1through C9. For several steps in the activation process, the product isan enzyme that catalyzes the subsequent step. This cascade providesamplification and activation of large amounts of complement by arelatively small initial signal.

The term “having CDC activity,” as used herein in reference to apolypeptide or protein, means that the polypeptide or protein (forexample, one comprising an immunoglobulin hinge region and animmunoglobulin constant region having CH2 and CH3 domains, such asderived from IgG (e.g., IgG1)) is capable of mediatingcomplement-dependent cytotoxicity (CDC) through binding of C1qcomplement protein and activation of the classical complement system.

“Redirected T-cell cytotoxicity” and “RTCC,” as used herein, refer to aT-cell-mediated process in which a cytotoxic T-cell is recruited to atarget cell using a multi-specific protein that is capable ofspecifically binding both the cytotoxic T-cell and the target cell, andwhereby a target-dependent cytotoxic T-cell response is elicited againstthe target cell.

The terms “neovascularization” and “angiogenesis” are usedinterchangeably herein. Neovascularization and angiogenesis refer to thegeneration of new blood vessels into cells, tissue, or organs. Thecontrol of angiogenesis is typically altered in certain disease statesand, in many case, the pathological damage associated with the diseaseis related to altered or unregulated angiogenesis. Persistant,unregulated angiogenesis occurs in a variety of disease states,including those characterized by the abnormal growth by endothelialcells, and supports the pathological damage seen in these conditionsincluding leakage and permeability of blood vessels.

The term “neovascular disorder” are used herein refers to any disease ordisorder having a pathology that is mediated, at least in part, byincreased or unregulated angiogenesis activity. Examples of suchdiseases or disorders include various cancers comprising solid tumors.Such diseases or disorders comprising a vasculature characterized byPSMA overexpression (e.g., certain cancers comprising solid tumors, suchas clear cell renal carcinoma, colorectal cancer, bladder cancer, andlung cancer) are particularly amenable to certain treatment methods forinhibition angiogenesis, as described further herein.

As used herein, the term “treatment,” “treating,” or “ameliorating”refers to either a therapeutic treatment or prophylactic/preventativetreatment. A treatment is therapeutic if at least one symptom of diseasein an individual receiving treatment improves or a treatment can delayworsening of a progressive disease in an individual, or prevent onset ofadditional associated diseases.

As used herein, the term “therapeutically effective amount (or dose)” or“effective amount (or dose)” of a specific binding molecule or compoundrefers to that amount of the compound sufficient to result inamelioration of one or more symptoms of the disease being treated in astatistically significant manner. When referring to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When referring to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredserially or simultaneously (in the same formuation or concurrently inseparate formulations).

As used herein, the term “transformation,” “transfection,” and“transduction” refer to the transfer of nucleic acid (i.e., a nucleotidepolymer) into a cell. As used herein, the term “genetic transformation”refers to the transfer and incorporation of DNA, especially recombinantDNA, into a cell. The transferred nucleic acid can be introduced into acell via an expression vector.

As used herein, the term “variant” or “variants” refers to a nucleicacid or polypeptide differing from a reference nucleic acid orpolypeptide, but retaining essential properties thereof. Generally,variants are overall closely similar, and, in many regions, identical tothe reference nucleic acid or polypeptide. For instance, a variant mayexhibit at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98% or at least about 99% sequence identity compared to the activeportion or full length reference nucleic acid or polypeptide.

The terms “light chain variable region” (also referred to as “lightchain variable domain” or “VL”) and “heavy chain variable region” (alsoreferred to as “heavy chain variable domain” or “VH”) refer to thevariable binding region from an antibody light and heavy chain,respectively. The variable binding regions are made up of discrete,well-defined sub-regions known as “complementarity determining regions”(CDRs) and “framework regions” (FRs). In one embodiment, the FRs arehumanized. The term “CL” refers to an “immunoglobulin light chainconstant region” or a “light chain constant region,” i.e., a constantregion from an antibody light chain. The term “CH” refers to an“immunoglobulin heavy chain constant region” or a “heavy chain constantregion,” which is further divisible, depending on the antibody isotypeinto CH1, CH2, and CH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4domains (IgE, IgM). A “Fab” (fragment antigen binding) is the part of anantibody that binds to antigens and includes the variable region and CH1domain of the heavy chain linked to the light chain via an inter-chaindisulfide bond.

III. PSMA-Binding Polypeptides, Proteins, and Components Thereof

The present disclosure provides polypeptides and proteins comprisingbinding domains, in particular, a first binding domain that specificallybinds PSMA. The polypeptides and proteins comprising binding domains ofthis disclosure can further comprise immunoglobulin constant regions,linker peptides, hinge regions, immunoglobulindimerization/heterodimerization domains, junctional amino acids, tags,etc. These components of the disclosed polypeptides and proteins aredescribed in further detail below.

Additionally, the PSMA-binding polypeptides and proteins disclosedherein can be in the form of an antibody or a fusion protein of any of avariety of different formats (e.g., the fusion protein can be in theform of a SMIP molecule, a PIMS molecule, a SCORPION molecule or anInterceptor molecule).

A PSMA-binding protein in accordance with the present inventiongenerally includes at least one PSMA-binding polypeptide chaincomprising (a) a PSMA-binding domain as set forth herein. In certainvariations, the PSMA-binding polypeptide further includes (b) a hingeregion carboxyl-terminal to the PSMA-binding domain, and (c) animmunoglobulin constant region (e.g., a SMIP molecule). In furthervariations, the PSMA-binding polypeptide further includes (d) a secondhinge region carboxyl-terminal to the immunoglobulin constant region,and (e) a second binding domain carboxyl-terminal to the second hingeregion (e.g., a SCORPION polypeptide).

In yet other variations, the PSMA-binding polypeptide comprises (b) ahinge region amino-terminal to the PSMA-binding domain, and (c) animmunoglobulin sub-region amino-terminal to the hinge region (e.g., aPIMS polypeptide).

Typically, PSMA-binding polypeptides of the above formats (SMIP,SCORPION, or PIMS) are capable of homodimerization, typically throughdisulfide bonding, via the immunoglobulin constant region and/or hingeregion (e.g., via an immunoglobulin constant region comprising IgG CH2and CH3 domains and an IgG hinge region). Thus, in certain embodimentsof the present invention, two identical PSMA-binding polypeptideshomodimerize to form a dimeric PSMA-binding protein.

In other embodiments, a PSMA-binding polypeptide further includes aheterodimerization domain that is capable of heterodimerization with adifferent heterodimerization domain in a second, non-identicalpolypeptide chain. In certain variations, the second polypeptide chainfor heterodimerization includes a second binding domain. Accordingly, incertain embodiments of the present invention, two non-identicalpolypeptide chains, one comprising the PSMA-binding domain and thesecond optionally comprising a second binding domain, dimerize to form aheterodimeric PSMA-binding protein.

PSMA-binding polypeptides, proteins, and their various components arefurther described herein below.

A. Binding Domains

As indicated above, an immunoglobulin binding polypeptide of the presentdisclosure comprises a binding domain that specifically binds PSMA. Insome variations, the PSMA-binding domain is capable of competing forbinding to PSMA with an antibody having V_(L) and V_(H) regions havingamino acid sequences as shown in SEQ ID NO:5 and SEQ ID NO:2,respectively (e.g., mAb 107-1A41, or with a single-chain Fv (scFv)having an amino acid sequence as shown in SEQ ID NO:21. In certainembodiments, the PSMA-binding domain comprises (i) an immunoglobulinlight chain variable region (V_(L)) comprising CDRs LCDR1, LCDR2, andLCDR3, and (ii) an immunoglobulin heavy chain variable region (V_(H))comprising CDRs HCDR1, HCDR2, and HCDR3. Suitable PSMA-binding domainsinclude those having V_(L) and V_(H) regions derived from mAb 107-1A4.In some such embodiments, LCDR3 has the amino acid sequence set forth inSEQ ID NO:17 and/or HCDR3 has the amino acid sequence set forth in SEQID NO:11; and LCDR1 and LCDR2 optionally have the amino acid sequencesas set forth in SEQ ID NO:15 and SEQ ID NO:16, respectively, and HCDR1and HCDR2 optionally have the amino acid sequences as set forth in SEQID NO:9 and SEQ ID NO:10, respectively. In some embodiments, forexample, LCDR1, LCDR2, and LCDR3 have the amino acid sequencesrespectively shown in SEQ ID NOs:15, 16, and 17; and/or HCDR1, HCDR2,and HCDR3 have the amino acid sequences as respectively shown in SEQ IDNOs:9, 10, and 11.

In certain embodiments, a PSMA-binding protein can comprise one or moreadditional binding domains (e.g., second binding domain) that bind atarget other than PSMA. These other target molecules can comprise, forexample, a particular cytokine or a molecule that targets the bindingdomain polypeptide to a particular cell type, a toxin, an additionalcell receptor, an antibody, etc.

In certain embodiments, a binding domain, for instance, as part of anInterceptor or SCORPION molecule, can comprise a TCR binding domain forrecruitment of T cells to target cells expressing PSMA. In certainembodiments, a polypeptide heterodimer as described herein can comprisea binding domain that specifically binds a TCR complex or a componentthereof (e.g., TCRα, TCRβ, CD3γ, CD3δ, and CD3ε) and another bindingdomain that specifically binds to PSMA.

Exemplary anti-CD3 antibodies from which the binding domain of thisdisclosure can be derived include CRIS-7 monoclonal antibody (Reinherz,E. L. et al. (eds.), Leukocyte typing II., Springer Verlag, New York,(1986); V_(L) and V_(H) amino acid sequences respectively shown in SEQID NO:153 (QVVLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITR) and SEQ IDNO:154 (QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNGFPYWGQ GTLVTVSA));HuM291 (Chau et al. (2001) Transplantion 71:941-950; V_(L) and V_(H)amino acid sequences respectively shown in SEQ ID NO:86(DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK) and SEQ ID NO:87 (QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSS)); BC3 monoclonal antibody (Anasetti etal. (1990) J. Exp. Med. 172:1691); OKT3 monoclonal antibody (Orthomulticenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) andderivatives thereof such as OKT3 ala-ala (also referred to as OKT3 AA-FLor OKT3 FL), a humanized, Fc variant with alanine substitutions atpositions 234 and 235 (Herold et al. (2003) J. Clin. Invest. 11:409);visilizumab (Carpenter et al. (2002) Blood 99:2712), G19-4 monoclonalantibody (Ledbetter et al., 1986, J. Immunol. 136:3945) and 145-2C11monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140: 3766). Anexemplary anti-TCR antibody is the BMA031 monoclonal antibody (Borst etal. (1990) Human Immunology 29:175-188).

In some embodiments, a binding domain is a single-chain Fv fragment(scFv) that comprises V_(H) and V_(L) regions specific for a target ofinterest. In certain embodiments, the V_(H) and V_(L) regions are human.

In certain embodiments, a PSMA-binding domain comprises or is a scFvthat is at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.5%, or 100% identical to an amino acid sequence of a scFvof SEQ ID NO: 19, 21, 30, 31, 34 or 35.

In related embodiments, a PSMA-binding domain comprises or is a sequencethat is at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.5%, or 100% identical to an amino acid sequence of alight chain variable region (V_(L)) (e.g., SEQ ID NO:23) or to a heavychain variable region (V_(H)) (e.g., SEQ ID NO:25 or SEQ ID NO:27), orboth.

In further embodiments, each CDR comprises no more than one, two, orthree substitutions, insertions or deletions, as compared to that from amonoclonal antibody or fragment or derivative thereof that specificallybinds to a target of interest (e.g., PSMA).

In some embodiments of a PSMA-binding protein comprising a secondbinding domain that specifically binds CD3ε, the second binding domaincompetes for binding to CD3ε with the CRIS-7 or HuM291 monoclonalantibody. In certain variations, the CD3-binding domain comprises animmunoglobulin light chain variable region (V_(L)) and an immunoglobulinheavy chain variable region (V_(H)) derived from the CRIS-7 or HuM291monoclonal antibody (e.g., the V_(L) and V_(H) of the second bindingdomain can be humanized variable regions comprising, respectively, thelight chain CDRs and the heavy chain CDRs of the monoclonal antibody).For example, the V_(L) and V_(H) regions derived from CRIS-7 can beselected from (a) a V_(L) region comprising an amino acid sequence thatis at least 95% identical or 100% to the amino acid sequence set forthin residues 139-245 of SEQ ID NO:47 and a V_(H) region comprising anamino acid sequence that is at least 95% identical or 100% to the aminoacid sequence set forth in residues 1-122 of SEQ ID NO:47; and (b) aV_(L) region comprising an amino acid sequence that is at least 95%identical or 100% identical to the amino acid sequence set forth inresidues 634-740 of SEQ ID NO:78 and a V_(H) region comprising an aminoacid sequence that is at least 95% or 100% identical to the amino acidsequence set forth in residues 496-616 of SEQ ID NO:78.

In certain embodiments, a binding domain V_(L) and/or V_(H) region ofthe present disclosure is derived from a V_(L) and/or V_(H) of a knownmonoclonal antibody (e.g., 107-1A4, CRIS-7, or HuM291) and containsabout one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions,about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions,about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acidsubstitutions (e.g., conservative amino acid substitutions ornon-conservative amino acid substitutions), or a combination of theabove-noted changes, when compared with the V_(L) and/or V_(H) of aknown monoclonal antibody. The insertion(s), deletion(s) orsubstitution(s) can be anywhere in the V_(L) and/or V_(H) region,including at the amino- or carboxyl-terminus or both ends of thisregion, provided that each CDR comprises zero changes or at most one,two, or three changes and provided a binding domain containing themodified V_(L) and/or V_(H) region can still specifically bind itstarget with an affinity similar to the wild type binding domain.

In some variations, the binding domain is a single-chain Fv (scFv)comprising immunoglobulin V_(L) and V_(H) regions joined by a peptidelinker. The use of peptide linkers for joining V_(L) and V_(H) regionsis well-known in the art, and a large number of publications existwithin this particular field. A widely used peptide linker is a 15merconsisting of three repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence((Gly₄Ser)₃) (SEQ ID NO:152). Other linkers have been used, and phagedisplay technology, as well as selective infective phage technology, hasbeen used to diversify and select appropriate linker sequences (Tang etal., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al., ProteinEng. 11, 405-410, 1998). In certain embodiments, the V_(L) and V_(H)regions are joined by a peptide linker having an amino acid sequencecomprising the formula (Gly₄Ser)_(n), wherein n=1-5 (SEQ ID NO:165).Other suitable linkers can be obtained by optimizing a simple linker(e.g., (Gly₄Ser)_(n)) through random mutagenesis.

In certain embodiments, a binding domain comprises humanizedimmunoglobulin V_(L) and/or V_(H) regions. Techniques for humanizingimmunoglobulin V_(L) and V_(H) regions are known in the art and arediscussed, for example, in United States Patent Application PublicationNo. 2006/0153837.

“Humanization” is expected to result in an antibody that is lessimmunogenic, with complete retention of the antigen-binding propertiesof the original molecule. In order to retain all of the antigen-bindingproperties of the original antibody, the structure of its antigenbinding site should be reproduced in the “humanized” version. This canbe achieved by grafting only the nonhuman CDRs onto human variableframework domains and constant regions, with or without retention ofcritical framework residues (Jones et al., Nature 321:522 (1986);Verhoeyen et al., Science 239:1539 (1988)) or by recombining the entirenonhuman variable domains (to preserve ligand-binding properties), but“cloaking” them with a human-like surface through judicious replacementof exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol.28:489 (1991)).

Essentially, humanization by CDR grafting involves recombining only theCDRs of a non-human antibody onto a human variable region framework anda human constant region. Theoretically, this should substantially reduceor eliminate immunogenicity (except if allotypic or idiotypicdifferences exist). However, it has been reported that some frameworkresidues of the original antibody also may need to be preserved(Reichmann et al., Nature, 332:323 (1988); Queen et al., Proc. Natl.Acad. Sci. USA, 86:10,029 (1989)).

The framework residues that need to be preserved are amenable toidentification through computer modeling. Alternatively, criticalframework residues can potentially be identified by comparing knownantigen-binding site structures (Padlan, Molec. Immunol., 31(3):169-217(1994), incorporated herein by reference).

The residues that potentially affect antigen binding fall into severalgroups. The first group comprises residues that are contiguous with theantigen site surface, which could therefore make direct contact withantigens. These residues include the amino-terminal residues and thoseadjacent to the CDRs. The second group includes residues that couldalter the structure or relative alignment of the CDRs, either bycontacting the CDRs or another peptide chain in the antibody. The thirdgroup comprises amino acids with buried side chains that could influencethe structural integrity of the variable domains. The residues in thesegroups are usually found in the same positions (Padlan, 1994, supra)although their positions as identified may differ depending on thenumbering system (see Kabat et al., “Sequences of proteins ofimmunological interest, 5th ed., Pub. No. 91-3242, U.S. Dept. Health &Human Services, NIH, Bethesda, Md., 1991).

Although the embodiments described herein involve the humanization ofSMIP, SCORPION, and Interceptor molecules, and not antibodies, knowledgeabout humanized antibodies in the art is applicable to the polypeptidesaccording to the invention.

B. Hinge Region

In certain embodiments, a hinge is a wild-type human immunoglobulinhinge region. In certain other embodiments, one or more amino acidresidues can be added at the amino- or carboxyl-terminus of a wild typeimmunoglobulin hinge region as part of a fusion protein constructdesign. For example, additional junction amino acid residues at thehinge amino-terminus can be “RT,” “RSS,” “TG,” or “T,” or at the hingecarboxyl-terminus can be “SG”, or a hinge deletion can be combined withan addition, such as ΔP with “SG” added at the carboxyl-terminus.

In certain embodiments, a hinge is an altered immunoglobulin hinge inwhich one or more cysteine residues in a wild type immunoglobulin hingeregion is substituted with one or more other amino acid residues (e.g.,serine or alanine).

Exemplary altered immunoglobulin hinges include an immunoglobulin humanIgG1 hinge region having one, two or three cysteine residues found in awild type human IgG1 hinge substituted by one, two or three differentamino acid residues (e.g., serine or alanine). An altered immunoglobulinhinge can additionally have a proline substituted with another aminoacid (e.g., serine or alanine). For example, the above-described alteredhuman IgG1 hinge can additionally have a proline locatedcarboxyl-terminal to the three cysteines of wild type human IgG1 hingeregion substituted by another amino acid residue (e.g., serine,alanine). In one embodiment, the prolines of the core hinge region arenot substituted.

In certain embodiments, a hinge polypeptide comprises or is a sequencethat is at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a wild type immunoglobulin hinge region, such as a wildtype human IgG1 hinge, a wild type human IgG2 hinge, or a wild typehuman IgG4 hinge.

In further embodiments, a hinge present in a PSMA-binding polypeptidecan be a hinge that is not based on or derived from an immunoglobulinhinge (i.e., not a wild-type immunoglobulin hinge or an alteredimmunoglobulin hinge). Examples for such hinges include peptides ofabout five to about 150 amino acids derived from an interdomain regionof a transmembrane protein or stalk region of a type II C-lectin, forinstance, peptides of about eight to 25 amino acids and peptides ofabout seven to 18 amino acids.

In certain embodiments, interdomain or stalk region hinges have seven to18 amino acids and can form an α-helical coiled coil structure. Incertain embodiments, interdomain or stalk region hinges contain 0, 1, 2,3, or 4 cysteines. Exemplary interdomain or stalk region hinges arepeptide fragments of the interdomain or stalk regions, such as ten to150 amino acid fragments from the stalk regions of CD69, CD72, CD94,NKG2A and NKG2D.

In certain embodiments, hinge sequences have about 5 to 150 amino acids,5 to 10 amino acids, 10 to 20 amino acids, 20 to 30 amino acids, 30 to40 amino acids, 40 to 50 amino acids, 50 to 60 amino acids, 5 to 60amino acids, 5 to 40 amino acids, 8 to 20 amino acids, or 10 to 15 aminoacids. The hinge can be primarily flexible, but can also provide morerigid characteristics or can contain primarily α-helical structure withminimal β-sheet structure. The lengths or the sequences of the hingescan affect the binding affinities of the binding domains to which thehinges are directly or indirectly (via another region or domain, such asan heterodimerization domain) connected as well as one or moreactivities of the Fc region portions to which the hinges are directly orindirectly connected.

In certain embodiments, hinge sequences are stable in plasma and serumand are resistant to proteolytic cleavage. The first lysine in the IgG1upper hinge region can be mutated to minimize proteolytic cleavage, forinstance, the lysine can be substituted with methionine, threonine,alanine or glycine, or is deleted.

In some embodiments of the invention, the PSMA-binding polypeptide iscapable of forming a heterodimer with a second polypeptide chain andcomprises a hinge region (a) immediately amino-terminal to animmunoglobulin constant region (e.g., amino-terminal to a CH2 domainwherein the immungobloubolin constant region includes CH2 and CH3domains, or amino-terminal to a CH3 domain wherein the immunoglobulinsub-regions includes CH3 and CH4 domains), (b) interposed between andconnecting a binding domain (e.g., scFv) and a immunoglobulinheterodimerization domain, (c) interposed between and connecting aimmunoglobulin heterodimerization domain and an immunoglobulin constantregion (e.g., wherein the immunoglobulin constant region includes CH2and CH3 domains or CH3 and CH4 domains), (d) interposed between andconnecting an immunoglobulin constant region and a binding domain, (e)at the amino-terminus of a polypeptide chain, or (f) at thecarboxyl-terminus of a polypeptide chain. A polypeptide chain comprisinga hinge region as described herein will be capable of associating with adifferent polypeptide chain to form a heterodimeric protein providedherein, and the heterodimer formed will contain a binding domain thatretains its target specificity or its specific target binding affinity.

In certain embodiments, a hinge present in a polypeptide that forms aheterodimer with another polypeptide chain can be an immunoglobulinhinge, such as a wild-type immunoglobulin hinge region or an alteredimmunoglobulin hinge region thereof. In certain embodiments, a hinge ofone polypeptide chain of a heterodimeric protein is identical to acorresponding hinge of the other polypeptide chain of the heterodimer.In certain other embodiments, a hinge of one chain is different fromthat of the other chain (in their length or sequence). The differenthinges in the different chains allow different manipulation of thebinding affinities of the binding domains to which the hinges areconnected, so that the heterodimer is able to preferentially bind to thetarget of one binding domain over the target of the other bindingdomain. For example, in certain embodiments, a heterodimeric protein hasa CD3- or TCR-binding domain in one chain and a PSMA-binding domain inanother chain. Having two different hinges in the two chains may allowthe heterodimer to bind to the PSMA first, and then to a CD3 or otherTCR component second. Thus, the heterodimer may recruit CD3⁺ T cells toPSMA-expressing cells (e.g., PSMA-expressing tumor cells), which in turnmay damage or destroy the PSMA-expressing cells.

Exemplary hinge regions suitable for use in accordance with the presentinvention are shown in the Tables 1 and 2 below. Additional exemplaryhinge regions are set forth in SEQ ID NOs: 241-244, 601, 78, 763-791,228, 379-434, 618-749 of WO2011/090762 (said sequences incorporated byreference herein).

TABLE 1 Exemplary hinge regions Hinge Region Amino Acid SequenceSEQ ID NO sss(s)-hIgG1 hinge EPKSSDKTHTSPPSS SEQ ID NO: 88csc(s)-hIgG1 hinge EPKSCDKTHTSPPCS SEQ ID NO: 89 ssc(s)-hIgG1 hingeEPKSSDKTHTSPPCS SEQ ID NO: 90 scc(s)-hIgG1 hinge EPKSSDKTHTCPPCSSEQ ID NO: 91 css(s)-hIgG1 hinge EPKSCDKTHTSPPSS SEQ ID NO: 92scs(s)-hIgG1 hinge EPKSSDKTHTCPPSS SEQ ID NO: 93 ccc(s)-hIgG1 hingeEPKSCDKTHTSPPCS SEQ ID NO: 94 ccc(p)-hIgG1 hinge EPKSCDKTHTSPPCPSEQ ID NO: 95 sss(p)-hIgG1 hinge EPKSSDKTHTSPPSP SEQ ID NO: 96csc(p)-hIgG1 hinge EPKSCDKTHTSPPCP SEQ ID NO: 97 ssc(p)-hIgG1 hingeEPKSSDKTHTSPPCP SEQ ID NO: 98 scc(p)-hIgG1 hinge EPKSSDKTHTCPPCPSEQ ID NO: 99 css(p)-hIgG1 hinge EPKSCDKTHTSPPSP SEQ ID NO: 100scs(p)-hIgG1 hinge EPKSSDKTHTCPPSP SEQ ID NO: 101 Scppcp SCPPCPSEQ ID NO: 102 STD1 NYGGGGSGGGGSGGGGSGNS SEQ ID NO: 103 STD2NYGGGGSGGGGSGGGGSGNY SEQ ID NO: 104 GGGGSGGGGSGGGGSGNS H1 NSSEQ ID NO: 105 H2 GGGGSGNS SEQ ID NO: 106 H3 NYGGGGSGNS SEQ ID NO: 107H4 GGGGSGGGGSGNS SEQ ID NO: 108 H5 NYGGGGSGGGGSGNS SEQ ID NO: 109 H6GGGGSGGGGSGGGGSGNS SEQ ID NO: 110 H7 GCPPCPNS SEQ ID NO: 62 (G₄S)₃GGGGSGGGGSGGGGS SEQ ID NO: 111 H105 SGGGGSGGGGSGGGGS SEQ ID NO: 155(G₄S)₄ GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 112 H75 (NKG2AQRHNNSSLNTGTQMAGHSPNS SEQ ID NO: 63 quadruple mutant) H83 (NKG2ASSLNTGTQMAGHSPNS SEQ ID NO: 65 derived) H106 (NKG2A QRHNNSSLNTGTQMAGHSSEQ ID NO: 156 derived) H81 (NKG2D EVQIPLTESYSPNS SEQ ID NO: 64 derived)H91 (NKG2D NSLANQEVQIPLTESYSPNS SEQ ID NO: 66 derived) H94SGGGGSGGGGSGGGGSPNS SEQ ID NO: 67

TABLE 2 Exemplary hinge regions (derived from H7 hinge, stalk region of a type II C-lectin, or interdomain region of a type Itransmembrane protein) Molecule and/or Hinge hinge from RegionAmino Acid Sequence which derived SEQ ID NO: H16 LSVKADFLTPSIGNS CD80SEQ ID NO: 113 H17 LSVKADFLTPSISCPPCPNS CD80 + H7 SEQ ID NO: 114 H18LSVLANFSQPEIGNS CD86 SEQ ID NO: 115 H19 LSVLANFSQPEISCPPCPNS CD86 + H7SEQ ID NO: 116 H20 LKIQERVSKPKISNS CD2 SEQ ID NO: 117 H21LKIQERVSKPKISCPPCPNS CD2  + H7 SEQ ID NO: 118 H22 LNVSERPFPPHIQNS CD22SEQ ID NO: 119 H23 LDVSERPFPPHIQSCPPCPNS CD22 + H7 SEQ ID NO: 120 H24REQLAEVTLSLKANS CD80 SEQ ID NO: 121 H25 REQLAEVTLSLKACPPCPNS CD80 + H7SEQ ID NO: 122 H26 RIHQMNSELSVLANS CD86 SEQ ID NO: 123 H27RIHQMNSELSVLACPPCPNS CD86 + H7 SEQ ID NO: 124 H28 DTKGKNVLEKIFSNS CD2SEQ ID NO: 125 H30 LPPETQESQEVTLNS CD22 SEQ ID NO: 126 H32RIHLNVSERPFPPNS CD22 SEQ ID NO: 127 H33 RIHLNVSERPFPPCPPCPNS CD22 + H7SEQ ID NO: 128 H36 GCPPCPGGGGSNS H7 SEQ ID NO: 129 H40 GCPPCPANS H7SEQ ID NO: 130 H41 GCPPCPANS H7 SEQ ID NO: 131 H42 GCPPCPNS H7SEQ ID NO: 132 H44 GGGASCPPCPGNS H7 SEQ ID NO: 133 H45 GGGASCPPCAGNS H7SEQ ID NO: 134 H46 GGGASCPPCANS H7 SEQ ID NO: 135 H47 LSVKADFLTPSIGNSCD80 SEQ ID NO: 136 H48 ADFLTPSIGNS CD80 SEQ ID NO: 137 H50LSVLANFSQPEIGNS CD86 SEQ ID NO: 138 H51 LSVLANFSQPEIGNS CD86SEQ ID NO: 139 H52 SQPEIVPISNS CD86 SEQ ID NO: 140 H53 SQPEIVPISCPPCPNSCD86 + H7 SEQ ID NO: 141 H54 SVLANFSQPEISCPPCPNS CD86 + H7SEQ ID NO: 142 H55 RIHQMNSELSVLANS CD86 SEQ ID NO: 143 H56 QMNSELSVLANSCD86 SEQ ID NO: 144 H57 VSERPFPPNS CD22 SEQ ID NO: 145 H58KPFFTCGSADTCPNS CD72 SEQ ID NO: 146 H59 KPFFTCGSADTCPNS CD72SEQ ID NO: 147 H60 QYNCPGQYTFSMPNS CD69 SEQ ID NO: 148 H61EPAFTPGPNIELQKDSDCPNS CD94 SEQ ID NO: 149 H62 QRHNNSSLNTRTQKARHCPNSNKG2A SEQ ID NO: 150 H63 NSLFNQEVQIPLTESYCPNS NKG2D SEQ ID NO: 151

C. Immunoglobulin Heterodimerization Domains

In certain embodiments, a PSMA-binding polypeptide or protein of theinvention can comprise an “immunoglobulin dimerization domain” or“immunoglobulin heterodimerization domain.”

An “immunoglobulin dimerization domain” or “immunoglobulinheterodimerization domain,” as used herein, refers to an immunoglobulindomain of a polypeptide chain that preferentially interacts orassociates with a different immunoglobulin domain of another polypeptidechain, wherein the interaction of the different immunoglobulinheterodimerization domains substantially contributes to or efficientlypromotes heterodimerization of the first and second polypeptide chains(i.e., the formation of a dimer between two different polypeptidechains, which is also referred to as a “heterodimer” or “heterodimericprotein”). The interactions between immunoglobulin heterodimerizationdomains “substantially contributes to or efficiently promotes” theheterodimerization of first and second polypeptide chains if there is astatistically significant reduction in the dimerization between thefirst and second polypeptide chains in the absence of the immunoglobulinheterodimerization domain of the first polypeptide chain and/or theimmunoglobulin heterodimerization domain of the second polypeptidechain. In certain embodiments, when the first and second polypeptidechains are co-expressed, at least 60%, at least about 60% to about 70%,at least about 70% to about 80%, at least 80% to about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second polypetpidechains form heterodimers with each other. Representative immunoglobulinheterodimerization domains include an immunoglobulin CH1 domain, animmunoglobulin CL1 domain (e.g., Cκ or Cλ isotypes), or derivativesthereof, including wild-type immunoglobulin CH1 and CL domains andaltered (or mutated) immunoglobulin CH1 and CL domains, such as providedherein.

Dimerization/heterodimerization domains can be used where it is desiredto form heterodimers from two non-identical polypeptide chains, whereone or both polypeptide chains comprises a binding domain. In certainembodiments, one polypeptide chain member of certain heterodimersdescribed herein does not contain a binding domain. As indicated above,a heterodimeric protein of the present disclosure comprises animmunoglobulin heterodimerization domain in each polypeptide chain. Theimmunoglobulin heterodimerization domains in the polypeptide chains of aheterodimer are different from each other and thus can be differentiallymodified to facilitate heterodimerization of both chains and to minimizehomodimerization of either chain. As shown in the examples,immunoglobulin heterodimerization domains provided herein allow forefficient heterodimerization between different polypeptides andfacilitate purification of the resulting heterodimeric protein.

As provided herein, immunoglobulin heterodimerization domains useful forpromoting heterodimerization of two different single chain polypeptides(e.g., one short and one long) according to the present disclosureinclude immunoglobulin CH1 and CL domains, for instance, human CH1 andCL domains. In certain embodiments, an immunoglobulin heterodimerizationdomain is a wild-type CH1 domain, such as a wild type IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domain. In further embodiments,an immunoglobulin heterodimerization domain is a wild-type human IgG1,IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domain as set forthin SEQ ID NOS:114, 186-192 and 194, respectively, of PCT Publication No.WO2011/090762 (said sequences incorporated by reference herein). Incertain embodiments, an immunoglobulin heterodimerization domain is awild-type human IgG1 CH1 domain as set forth in SEQ ID NO:114 ofWO2011/090762 (said sequence incorporated by reference herein).

In further embodiments, an immunoglobulin heterodimerization domain isan altered immunoglobulin CH1 domain, such as an altered IgG1, IgG2,IgG3, IgG4, IgA1, IgA2 IgD, IgE, or IgM CH1 domain. In certainembodiments, an immunoglobulin heterodimerization domain is an alteredhuman IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domain.In still further embodiments, a cysteine residue of a wild-type CH1domain (e.g., a human CH1) involved in forming a disulfide bond with awild type immunoglobulin CL domain (e.g., a human CL) is deleted orsubstituted in the altered immunoglobulin CH1 domain such that adisulfide bond is not formed between the altered CH1 domain and thewild-type CL domain.

In certain embodiments, an immunoglobulin heterodimerization domain is awild-type CL domain, such as a wild type Cκ domain or a wild type Cλdomain. In certain embodiments, an immunoglobulin heterodimerizationdomain is a wild type human Cκ or human Cλ domain as set forth in SEQ IDNOS:112 and 113, respectively, of WO2011/090762 (said sequencesincorporated by reference herein). In further embodiments, animmunoglobulin heterodimerization domain is an altered immunoglobulin CLdomain, such as an altered Cκ or Cλ domain, for instance, an alteredhuman Cκ or human Cλ domain.

In certain embodiments, a cysteine residue of a wild-type CL domain(e.g., a human CL) involved in forming a disulfide bond with a wild typeimmunoglobulin CH1 domain (e.g., a human CH1) is deleted or substitutedin the altered immunoglobulin CL domain. Such altered CL domains canfurther comprise an amino acid deletion at their amino-termini. Anexemplary Cκ domain is set forth in SEQ ID NO:141 of WO2011/090762 (saidsequence incorporated by reference herein), in which the first arginineand the last cysteine of the wild type human Ck domain are both deleted.In certain embodiments, only the last cysteine of the wild type human Ckdomain is deleted in the altered Ck domain because the first argininedeleted from the wild type human Ck domain can be provided by a linkerthat has an arginine at its carboxyl-terminus and links theamino-terminus of the altered Ck domain with another domain (e.g., animmunoglobulin sug-region, such as a sub-region comprisingimmunoglobulin CH2 and CH3 domains). An exemplary Cλ domain is set forthin SEQ ID NO:140 of WO2011/090762 (said sequence incorporated byreference herein), in which the first arginine of a wild type human Cλdomain is deleted and the cysteine involved in forming a disulfide bondwith a cysteine in a CH1 domain is substituted by a serine.

In further embodiments, an immunoglobulin heterodimerization domain isan altered Cκ domain that contains one or more amino acid substitutions,as compared to a wild type Cκ domain, at positions that may be involvedin forming the interchain-hydrogen bond network at a Cκ-Cκ interface.For example, in certain embodiments, an immunoglobulinheterodimerization domain is an altered human Cκ domain having one ormore amino acids at positions N29, N30, Q52, V55, T56, S68 or T70 thatare substituted with a different amino acid. The numbering of the aminoacids is based on their positions in the altered human Cκ sequence asset forth in SEQ ID NO:141 of WO2011/090762 (said sequence incorporatedby reference herein). In certain embodiments, an immunoglobulinheterodimerization domain is an altered human Cκ domain having one, two,three or four amino acid substitutions at positions N29, N30, V55, orT70. The amino acid used as a substitute at the above-noted positionscan be an alanine, or an amino acid residue with a bulk side chainmoiety such as arginine, tryptophan, tyrosine, glutamate, glutamine, orlysine. Additional amino acid residues that can be used to substituteamino acid residues of the wild type human Ck sequence at the abovenoted positions (e.g., N30) include aspartate, methionine, serine andphenyalanine. Exemplary altered human Cκ domains are set forth in SEQ IDNOS:142-178 of WO2011/090762 (said sequences incorporated by referenceherein). Altered human Cκ domains are those that facilitateheterodimerization with a CH1 domain, but minimize homodimerization withanother Cκ domain. Representative altered human Cκ domains are set forthin SEQ ID NOS:160 (N29W V55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29AN30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55AT70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176 (N29Y N30YV55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E), 838 (N30D V55AT70E), 839 (N30M V55A T70E), 840 (N30S V55A T70E), and 841 (N30F V55AT70E) of WO2011/090762 (said sequences incorporated by referenceherein).

In certain embodiments, in addition to or alternative to the mutationsin Ck domains described herein, both the immunoglobulinheterodimerization domains (i.e., immunoglobulin CH1 and CL domains) ofa polypeptide heterodimer have mutations so that the resultingimmunoglobulin heterodimerization domains form salt bridges (i.e., ionicinteractions) between the amino acid residues at the mutated sites. Forexample, the immunoglobulin heterodimerization domains of a polypeptideheterodimer can be a mutated CH1 domain in combination with a mutated Ckdomain. In the mutated CH1 domain, valine at position 68 (V68) of thewild type human CH1 domain is substituted by an amino acid residuehaving a negative charge (e.g., aspartate or glutamate), whereas leucineat position 29 (L29) of a mutated human Ck domain in which the firstarginine and the last cysteine have been deleted is substituted by anamino acid residue having a positive charge (e.g., lysine, arginine orhistidine). The charge-charge interaction between the amino acid residuehaving a negative charge of the resulting mutated CH1 domain and theamino acid residue having a positive charge of the resulting mutated Ckdomain forms a salt bridge, which stabilizes the heterodimeric interfacebetween the mutated CH1 and Ck domains. Alternatively, V68 of the wildtype CH1 can be substituted by an amino acid residue having a positivecharge, whereas L29 of a mutated human Ck domain in which the firstarginine and the last cysteine have been deleted can be substituted byan amino acid residue having a negative charge. Exemplary mutated CH1sequences in which V68 is substituted by an amino acid with either anegative or positive charge are set forth in SEQ ID NOS:844 and 845 ofWO2011/090762 (said sequences incorporated by reference herein).Exemplary mutated Ck sequences in which L29 is substituted by an aminoacid with either a negative or positive charge are set forth in SEQ IDNOS:842 and 843 of WO2011/090762 (said sequences incorporated byreference herein).

Positions other than V68 of human CH1 domain and L29 of human Ck domaincan be substituted with amino acids having opposite charges to produceionic interactions between the amino acids in addition or alternative tothe mutations in V68 of CH1 domain and L29 of Ck domain. Such positionscan be identified by any suitable method, including random mutagenesis,analysis of the crystal structure of the CH1-Ck pair to identify aminoacid residues at the CH1-Ck interface, and further identifying suitablepositions among the amino acid residues at the CH1-Ck interface using aset of criteria (e.g., propensity to engage in ionic interactions,proximity to a potential partner residue, etc.).

In certain embodiments, polypeptide heterodimers of the presentdisclosure contain only one pair of immunoglobulin heterodimerizationdomains. For example, a first chain of a polypeptide heterodimer cancomprise a CH1 domain as an immunoglobulin heterodimerization domain,while a second chain can comprise a CL domain (e.g., a Cκ or CA) as animmunoglobulin heterodimerization domain. Alternatively, a first chaincan comprise a CL domain (e.g., a Cκ or Cλ) as an immunoglobulinheterodimerization domain, while a second chain can comprise a CH1domain as an immunoglobulin heterodimerization domain. As set forthherein, the immunoglobulin heterodimerization domains of the first andsecond chains are capable of associating to form a heterodimeric proteinof this disclosure.

In certain other embodiments, heterodimeric proteins of the presentdisclosure can have two pairs of immunoglobulin heterodimerizationdomains. For example, a first chain of a heterodimer can comprise twoCH1 domains, while a second chain can have two CL domains that associatewith the two CH1 domains in the first chain. Alternatively, a firstchain can comprise two CL domains, while a second chain can have two CH1domains that associate with the two CL domains in the first chain. Incertain embodiments, a first polypeptide chain comprises a CH1 domainand a CL domain, while a second polypeptide chain comprises a CL domainand a CH1 domain that associate with the CH1 domain and the CL domain,respectively, of the first polypeptide chain.

In the embodiments where a heterodimeric protein comprises only oneheterodimerization pair (i.e., one immunoglobulin heterodimerizationdomain in each chain), the immunoglobulin heterodimerization domain ofeach chain can be located amino-terminal to the immunoglobulin constantregion of that chain. Alternatively, the immunoglobulinheterodimerization domain in each chain can be located carboxyl-terminalto the immunoglobulin constant region of that chain.

In the embodiments where a heterodimeric protein comprises twoheterodimerization pairs (i.e., two immunoglobulin heterodimerizationdomains in each chain), both immunoglobulin heterodimerization domainsin each chain can be located amino-terminal to the immunoglobulinconstant region of that chain. Alternatively, both immunoglobulinheterodimerization domains in each chain can be locatedcarboxyl-terminal to the immunoglobulin constant region of that chain.In further embodiments, one immunoglobulin heterodimerization domain ineach chain can be located amino-terminal to the immunoglobulin constantregion of that chain, while the other immunoglobulin heterodimerizationdomain of each chain can be located carboxyl-terminal to theimmunoglobulin constant region of that chain. In other words, in thoseembodiments, the immunoglobulin constant region is interposed betweenthe two immunoglobulin heterodimerization domains of each chain.

D. Immunoglobulin Constant Regions

As indicated herein, in certain embodiments, PSMA-binding polypeptidesof the present disclosure (e.g., SMIP, PIMS, SCORPION, and Interceptormolecules) comprise an immunoglobulin constant region (also referred toas an constant region) in each polypeptide chain. The inclusion of animmunoglobulin constant region slows clearance of the homodimeric andheterodimeric proteins formed from two PSMA-binding polypeptide chainsfrom circulation after administration to a subject. By mutations orother alterations, an immunoglobulin constant region further enablesrelatively easy modulation of dimeric polypeptide effector functions(e.g., ADCC, ADCP, CDC, complement fixation, and binding to Fcreceptors), which can either be increased or decreased depending on thedisease being treated, as known in the art and described herein. Incertain embodiments, an immunoglobulin constant region of one or both ofthe polypeptide chains of the polypeptide homodimers and heterodimers ofthe present disclosure will be capable of mediating one or more of theseeffector functions In other embodiments, one or more of these effectorfunctions are reduced or absent in an immunoglobulin constant region ofone or both of the polypeptide chains of the polypeptide homodimers andheterodimers of the present disclosure, as compared to a correspondingwild-type immunoglobulin constant region. For example, for dimericPSMA-binding polypeptides designed to elicit RTCC, such as, e.g., viathe inclusion of a CD3-binding domain, an immunoglobulin constant regionpreferably has reduced or no effector function relative to acorresponding wild-type immunoglobulin constant region.

An immunoglobulin constant region present in PSMA binding polypeptidesof the present disclosure can comprise of or is derived from part or allof: a CH2 domain, a CH3 domain, a CH4 domain, or any combinationthereof. For example, an immunoglobulin constant region can comprise aCH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4domains, two CH3 domains, a CH4 domain, two CH4 domains, and a CH2domain and part of a CH3 domain.

A CH2 domain that can form an immunoglobulin constant region of aPSMA-binding polypeptide of the present disclosure can be a wild typeimmunoglobulin CH2 domain or an altered immunoglobulin CH2 domainthereof from certain immunoglobulin classes or subclasses (e.g., IgG1,IgG2, IgG3, IgG4, IgA1, IgA2, or IgD) and from various species(including human, mouse, rat, and other mammals).

In certain embodiments, a CH2 domain is a wild type human immunoglobulinCH2 domain, such as wild type CH2 domains of human IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, or IgD, as set forth in SEQ ID NOS:115, 199-201 and195-197, respectively, of PCT Publication WO2011/090762 (said sequencesincorporated by reference herein). In certain embodiments, the CH2domain is a wild type human IgG1 CH2 domain as set forth in SEQ IDNO:115 of WO2011/090762 (said sequence incorporated by referenceherein).

In certain embodiments, a CH2 domain is an altered immunoglobulin CH2region (e.g., an altered human IgG1 CH2 domain) that comprises an aminoacid substitution at the asparagine of position 297 (e.g., asparagine toalanine). Such an amino acid substitution reduces or eliminatesglycosylation at this site and abrogates efficient Fc binding to FcγRand C1q. The sequence of an altered human IgG1 CH2 domain with an Asn toAla substitution at position 297 is set forth in SEQ ID NO:324 ofWO2011/090762 said (sequence incorporated by reference herein).

In certain embodiments, a CH2 domain is an altered immunoglobulin CH2region (e.g., an altered human IgG1 CH2 domain) that comprises at leastone substitution or deletion at positions 234 to 238. For example, animmunoglobulin CH2 region can comprise a substitution at position 234,235, 236, 237 or 238, positions 234 and 235, positions 234 and 236,positions 234 and 237, positions 234 and 238, positions 234-236,positions 234, 235 and 237, positions 234, 236 and 238, positions 234,235, 237, and 238, positions 236-238, or any other combination of two,three, four, or five amino acids at positions 234-238. In addition oralternatively, an altered CH2 region can comprise one or more (e.g.,two, three, four or five) amino acid deletions at positions 234-238, forinstance, at one of position 236 or position 237 while the otherposition is substituted. The above-noted mutation(s) decrease oreliminate the antibody-dependent cell-mediated cytotoxicity (ADCC)activity or Fc receptor-binding capability of a polypeptide heterodimerthat comprises the altered CH2 domain. In certain embodiments, the aminoacid residues at one or more of positions 234-238 has been replaced withone or more alanine residues. In further embodiments, only one of theamino acid residues at positions 234-238 have been deleted while one ormore of the remaining amino acids at positions 234-238 can besubstituted with another amino acid (e.g., alanine or serine).

In certain other embodiments, a CH2 domain is an altered immunoglobulinCH2 region (e.g., an altered human IgG1 CH2 domain) that comprises oneor more amino acid substitutions at positions 253, 310, 318, 320, 322,and 331. For example, an immunoglobulin CH2 region can comprise asubstitution at position 253, 310, 318, 320, 322, or 331, positions 318and 320, positions 318 and 322, positions 318, 320 and 322, or any othercombination of two, three, four, five or six amino acids at positions253, 310, 318, 320, 322, and 331. The above-noted mutation(s) decreaseor eliminate the complement-dependent cytotoxicity (CDC) of apolypeptide heterodimer that comprises the altered CH2 domain.

In certain other embodiments, in addition to the amino acid substitutionat position 297, an altered CH2 region (e.g., an altered human IgG1 CH2domain) can further comprise one or more (e.g., two, three, four, orfive) additional substitutions at positions 234-238. For example, animmunoglobulin CH2 region can comprise a substitution at positions 234and 297, positions 234, 235, and 297, positions 234, 236 and 297,positions 234-236 and 297, positions 234, 235, 237 and 297, positions234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions236-238 and 297, or any combination of two, three, four, or five aminoacids at positions 234-238 in addition to position 297. In addition oralternatively, an altered CH2 region can comprise one or more (e.g.,two, three, four or five) amino acid deletions at positions 234-238,such as at position 236 or position 237. The additional mutation(s)decreases or eliminates the antibody-dependent cell-mediatedcytotoxicity (ADCC) activity or Fc receptor-binding capability of apolypeptide heterodimer that comprises the altered CH2 domain. Incertain embodiments, the amino acid residues at one or more of positions234-238 have been replaced with one or more alanine residues. In furtherembodiments, only one of the amino acid residues at positions 234-238has been deleted while one or more of the remaining amino acids atpositions 234-238 can be substituted with another amino acid (e.g.,alanine or serine).

In certain embodiments, in addition to one or more (e.g., 2, 3, 4, or 5)amino acid substitutions at positions 234-238, a mutated CH2 region(e.g., an altered human IgG1 CH2 domain) in a fusion protein of thepresent disclosure can contain one or more (e.g., 2, 3, 4, 5, or 6)additional amino acid substitutions (e.g., substituted with alanine) atone or more positions involved in complement fixation (e.g., atpositions I253, H310, E318, K320, K322, or P331). Examples of mutatedimmunoglobulin CH2 regions include human IgG1, IgG2, IgG4 and mouseIgG2a CH2 regions with alanine substitutions at positions 234, 235, 237(if present), 318, 320 and 322. An exemplary mutated immunoglobulin CH2region is mouse IGHG2c CH2 region with alanine substitutions at L234,L235, G237, E318, K320, and K322.

In still further embodiments, in addition to the amino acid substitutionat position 297 and the additional deletion(s) or substitution(s) atpositions 234-238, an altered CH2 region (e.g., an altered human IgG1CH2 domain) can further comprise one or more (e.g., two, three, four,five, or six) additional substitutions at positions 253, 310, 318, 320,322, and 331. For example, an immunoglobulin CH2 region can comprise a(1) substitution at position 297, (2) one or more substitutions ordeletions or a combination thereof at positions 234-238, and one or more(e.g., 2, 3, 4, 5, or 6) amino acid substitutions at positions I253,H310, E318, K320, K322, and P331, such as one, two, three substitutionsat positions E318, K320 and K322. The amino acids at the above-notedpositions can be substituted by alanine or serine.

In certain embodiments, an immunoglobulin CH2 region polypeptidecomprises: (i) an amino acid substitution at the asparagines of position297 and one amino acid substitution at position 234, 235, 236 or 237;(ii) an amino acid substitution at the asparagine of position 297 andamino acid substitutions at two of positions 234-237; (iii) an aminoacid substitution at the asparagine of position 297 and amino acidsubstitutions at three of positions 234-237; (iv) an amino acidsubstitution at the asparagine of position 297, amino acid substitutionsat positions 234, 235 and 237, and an amino acid deletion at position236; (v) amino acid substitutions at three of positions 234-237 andamino acid substitutions at positions 318, 320 and 322; or (vi) aminoacid substitutions at three of positions 234-237, an amino acid deletionat position 236, and amino acid substitutions at positions 318, 320 and322.

Exemplary altered immunoglobulin CH2 regions with amino acidsubstitutions at the asparagine of position 297 include: human IgG1 CH2region with alanine substitutions at L234, L235, G237 and N297 and adeletion at G236 (SEQ ID NO:325 of WO2011/090762, said sequenceincorporated by reference herein), human IgG2 CH2 region with alaninesubstitutions at V234, G236, and N297 (SEQ ID NO:326 of WO2011/090762,said sequence incorporated by reference herein), human IgG4 CH2 regionwith alanine substitutions at F234, L235, G237 and N297 and a deletionof G236 (SEQ ID NO:322 of WO2011/090762, said sequence incorporated byreference herein), human IgG4 CH2 region with alanine substitutions atF234 and N297 (SEQ ID NO:343 of WO2011/090762, said sequenceincorporated by reference herein), human IgG4 CH2 region with alaninesubstitutions at L235 and N297 (SEQ ID NO:344 of WO2011/090762, saidsequence incorporated by reference herein), human IgG4 CH2 region withalanine substitutions at G236 and N297 (SEQ ID NO:345 of WO2011/090762,said sequence incorporated by reference herein), and human IgG4 CH2region with alanine substitutions at G237 and N297 (SEQ ID NO:346 ofWO2011/090762, said sequence incorporated by reference herein).

In certain embodiments, in addition to the amino acid substitutionsdescribed above, an altered CH2 region (e.g., an altered human IgG1 CH2domain) can contain one or more additional amino acid substitutions atone or more positions other than the above-noted positions. Such aminoacid substitutions can be conservative or non-conservative amino acidsubstitutions. For example, in certain embodiments, P233 can be changedto E233 in an altered IgG2 CH2 region (see, e.g., SEQ ID NO:326 ofWO2011/090762, said sequence incorporated by reference herein). Inaddition or alternatively, in certain embodiments, the altered CH2region can contain one or more amino acid insertions, deletions, orboth. The insertion(s), deletion(s) or substitution(s) can be anywherein an immunoglobulin CH2 region, such as at the N- or C-terminus of awild type immunoglobulin CH2 region resulting from linking the CH2region with another region (e.g., a binding domain or an immunoglobulinheterodimerization domain) via a hinge.

In certain embodiments, an altered CH2 region in a polypeptide of thepresent disclosure comprises or is a sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identical to a wildtype immunoglobulin CH2 region, such as the CH2 region of wild typehuman IgG1, IgG2, or IgG4, or mouse IgG2a (e.g., IGHG2c).

An altered immunoglobulin CH2 region in a PSMA-binding polypeptide ofthe present disclosure can be derived from a CH2 region of variousimmunoglobulin isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, andIgD, from various species (including human, mouse, rat, and othermammals). In certain embodiments, an altered immunoglobulin CH2 regionin a fusion protein of the present disclosure can be derived from a CH2region of human IgG1, IgG2 or IgG4, or mouse IgG2a (e.g., IGHG2c), whosesequences are set forth in SEQ ID NOS:115, 199, 201, and 320 ofWO2011/090762 (said sequences incorporated by reference herein).

In certain embodiments, an altered CH2 domain is a human IgG1 CH2 domainwith alanine substitutions at positions 235, 318, 320, and 322 (i.e., ahuman IgG1 CH2 domain with L235A, E318A, K320A and K322A substitutions)(SEQ ID NO:595 of WO2011/090762, said sequence incorporated by referenceherein), and optionally an N297 mutation (e.g., to alanine). In certainother embodiments, an altered CH2 domain is a human IgG1 CH2 domain withalanine substitutions at positions 234, 235, 237, 318, 320 and 322(i.e., a human IgG1 CH2 domain with L234A, L235A, G237A, E318A, K320Aand K322A substitutions) (SEQ ID NO:596 of WO2011/090762, said sequenceincorporated by reference herein), and optionally an N297 mutation(e.g., to alanine).

In certain embodiments, an altered CH2 domain is an altered human IgG1CH2 domain with mutations known in the art that enhance immunologicalactivities such as ADCC, ADCP, CDC, complement fixation, Fc receptorbinding, or any combination thereof.

The CH3 domain that can form an immunoglobulin constant region of aPSMA-binding polypeptide of the present disclosure can be a wild typeimmunoglobulin CH3 domain or an altered immunoglobulin CH3 domainthereof from certain immunoglobulin classes or subclasses (e.g., IgG1,IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM) of various species(including human, mouse, rat, and other mammals). In certainembodiments, a CH3 domain is a wild type human immunoglobulin CH3domain, such as wild type CH3 domains of human IgG1, IgG2, IgG3, IgG4,IgA1, IgA2, IgD, IgE, or IgM as set forth in SEQ ID NOS:116, 208-210,204-207, and 212, respectively of WO2011/090762 (said sequencesincorporated by reference herein). In certain embodiments, the CH3domain is a wild type human IgG1 CH3 domain as set forth in SEQ IDNO:116 of WO2011/090762 (said sequence incorporated by referenceherein). In certain embodiments, a CH3 domain is an altered humanimmunoglobulin CH3 domain, such as an altered CH3 domain based on orderived from a wild-type CH3 domain of human IgG1, IgG2, IgG3, IgG4,IgA1, IgA2, IgD, IgE, or IgM antibodies. For example, an altered CH3domain can be a human IgG1 CH3 domain with one or two mutations atpositions H433 and N434 (positions are numbered according to EUnumbering). The mutations in such positions can be involved incomplement fixation. In certain other embodiments, an altered CH3 domaincan be a human IgG1 CH3 domain but with one or two amino acidsubstitutions at position F405 or Y407. The amino acids at suchpositions are involved in interacting with another CH3 domain. Incertain embodiments, an altered CH3 domain can be an altered human IgG1CH3 domain with its last lysine deleted. The sequence of this alteredCH3 domain is set forth in SEQ ID NO:761 of WO2011/090762 (said sequenceincorporated by reference herein).

In certain embodiments, PSMA-binding polypeptides forming a polypeptideheterodimer comprise a CH3 pair that comprises so called“knobs-into-holes” mutations (see, Marvin and Zhu, Acta PharmacologicaSinica 26:649-58, 2005; Ridgway et al., Protein Engineering 9:617-21,1966). More specifically, mutations can be introduced into each of thetwo CH3 domains of each polypeptide chain so that the stericcomplementarity required for CH3/CH3 association obligates these two CH3domains to pair with each other. For example, a CH3 domain in one singlechain polypeptide of a polypeptide heterodimer can contain a T366Wmutation (a “knob” mutation, which substitutes a small amino acid with alarger one), and a CH3 domain in the other single chain polypeptide ofthe polypeptide heterodimer can contain a Y407A mutation (a “hole”mutation, which substitutes a large amino acid with a smaller one).Other exemplary knobs-into-holes mutations include (1) a T366Y mutationin one CH3 domain and a Y407T in the other CH3 domain, and (2) a T366Wmutation in one CH3 domain and T366S, L368A and Y407V mutations in theother CH3 domain.

The CH4 domain that can form an immunoglobulin constant region ofPSMA-binding polypeptides of the present disclosure can be a wild typeimmunoglobulin CH4 domain or an altered immunoglobulin CH4 domainthereof from IgE or IgM molecules. In certain embodiments, the CH4domain is a wild type human immunoglobulin CH4 domain, such as wild typeCH4 domains of human IgE and IgM molecules as set forth in SEQ IDNOS:213 and 214, respectively, of WO2011/090762 (said sequencesincorporated by reference herein). In certain embodiments, a CH4 domainis an altered human immunoglobulin CH4 domain, such as an altered CH4domain based on or derived from a CH4 domain of human IgE or IgMmolecules, which have mutations that increase or decrease animmunological activity known to be associated with an IgE or IgM Fcregion.

In certain embodiments, an immunoglobulin constant region of PSMAbinding polypeptides of the present disclosure comprises a combinationof CH2, CH3 or CH4 domains (i.e., more than one constant region domainselected from CH2, CH3 and CH4). For example, the immunoglobulinconstant region can comprise CH2 and CH3 domains or CH3 and CH4 domains.In certain other embodiments, the immunoglobulin constant region cancomprise two CH3 domains and no CH2 or CH4 domains (i.e., only two ormore CH3). The multiple constant region domains that form animmunoglobulin constant region can be based on or derived from the sameimmunoglobulin molecule, or the same class or subclass immunoglobulinmolecules. In certain embodiments, the immunoglobulin constant region isan IgG CH2CH3 (e.g., IgG1 CH2CH3, IgG2 CH2CH3, and IgG4 CH2CH3) and canbe a human (e.g., human IgG1, IgG2, and IgG4) CH2CH3. For example, incertain embodiments, the immunoglobulin constant region comprises (1)wild type human IgG1 CH2 and CH3 domains, (2) human IgG1 CH2 with N297Asubstitution (i.e., CH2(N297A)) and wild type human IgG1 CH3, or (3)human IgG1 CH2(N297A) and an altered human IgG1 CH3 with the last lysinedeleted.

Alternatively, the multiple constant region domains can be based on orderived from different immunoglobulin molecules, or different classes orsubclasses immunoglobulin molecules. For example, in certainembodiments, an immunoglobulin constant region comprises both human IgMCH3 domain and human IgG1 CH3 domain. The multiple constant regiondomains that form an immunoglobulin constant region can be directlylinked together or can be linked to each other via one or more (e.g.,about 2-10) amino acids.

Exemplary immunoglobulin constant regions are set forth in SEQ IDNOS:305-309, 321, 323, 341, 342, and 762 of WO2011/090762 (saidsequences incorporated by reference herein).

In certain embodiments, the immunoglobulin constant regions of bothPSMA-binding polypeptides of a polypeptide homodimer or heterodimer areidentical to each other. In certain other embodiments, theimmunoglobulin constant region of one polypeptide chain of aheterodimeric protein is different from the immunoglobulin constantregion of the other polypeptide chain of the heterodimer. For example,one immunoglobulin constant region of a heterodimeric protein cancontain a CH3 domain with a “knob” mutation, whereas the otherimmunoglobulin constant region of the heterodimeric protein can containa CH3 domain with a “hole” mutation.

IV. Nucleic Acids, Host Cells, and Methods for Production

The invention also includes nucleic acids (e.g., DNA or RNA) encoding aPSMA-binding polypeptide as described herein, or one or more polypeptidechains of a dimeric or heterodimeric PSMA-binding protein as describedherein. Nucleic acids of the invention include nucleic acids having aregion that is substantially identical to a polynucleotide as listed inTable 3, infra. In certain embodiments, a nucleic acid in accordancewith the present invention has at least 80%, typically at least about90%, and more typically at least about 95% or at least about 98%identity to a polypeptide-encoding polynucleotide as listed in Table 3.Nucleic acids of the invention also include complementary nucleic acids.In some instances, the sequences will be fully complementary (nomismatches) when aligned. In other instances, there can be up to about a20% mismatch in the sequences. In some embodiments of the invention areprovided nucleic acids encoding both first and second polypeptide chainsof a heterodimeric PSMA-binding protein of the invention. The nucleicacid sequences provided herein can be exploited using codonoptimization, degenerate sequence, silent mutations, and other DNAtechniques to optimize expression in a particular host, and the presentinvention encompasses such sequence modifications.

Polynucleotide molecules comprising a desired polynucleotide sequenceare propagated by placing the molecule in a vector. Viral and non-viralvectors are used, including plasmids. The choice of plasmid will dependon the type of cell in which propagation is desired and the purpose ofpropagation. Certain vectors are useful for amplifying and making largeamounts of the desired DNA sequence. Other vectors are suitable forexpression in cells in culture. Still other vectors are suitable fortransfer and expression in cells in a whole animal or person. The choiceof appropriate vector is well within the skill of the art. Many suchvectors are available commercially. The partial or full-lengthpolynucleotide is inserted into a vector typically by means of DNAligase attachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

For expression, an expression cassette or system may be employed. Toexpress a nucleic acid encoding a polypeptide disclosed herein, anucleic acid molecule encoding the polypeptide, operably linked toregulatory sequences that control transcriptional expression in anexpression vector, is introduced into a host cell. In addition totranscriptional regulatory sequences, such as promoters and enhancers,expression vectors can include translational regulatory sequences and amarker gene which is suitable for selection of cells that carry theexpression vector. The gene product encoded by a polynucleotide of theinvention is expressed in any convenient expression system, including,for example, bacterial, yeast, insect, amphibian and mammalian systems.In the expression vector, the polypeptide-encoding polynucleotide islinked to a regulatory sequence as appropriate to obtain the desiredexpression properties. These can include promoters, enhancers,terminators, operators, repressors, and inducers. The promoters can beregulated (e.g., the promoter from the steroid inducible pIND vector(Invitrogen)) or constitutive (e.g., promoters from CMV, SV40,Elongation Factor, or LTR sequences). These are linked to the desirednucleotide sequence using the techniques described above for linkage tovectors. Any techniques known in the art can be used. Accordingly, theexpression vector will generally provide a transcriptional andtranslational initiation region, which can be inducible or constitutive,where the coding region is operably linked under the transcriptionalcontrol of the transcriptional initiation region, and a transcriptionaland translational termination region.

An expression cassette (“expression unit”) can be introduced into avariety of vectors, e.g., plasmid, BAC, YAC, bacteriophage such aslambda, P1, M13, etc., plant or animal viral vectors (e.g.,retroviral-based vectors, adenovirus vectors), and the like, where thevectors are normally characterized by the ability to provide selectionof cells comprising the expression vectors. The vectors can provide forextrachromosomal maintenance, particularly as plasmids or viruses, orfor integration into the host chromosome. Where extrachromosomalmaintenance is desired, an origin sequence is provided for thereplication of the plasmid, which can be low- or high copy-number. Awide variety of markers are available for selection, particularly thosewhich protect against toxins, more particularly against antibiotics. Theparticular marker that is chosen is selected in accordance with thenature of the host, where in some cases, complementation can be employedwith auxotrophic hosts. Introduction of the DNA construct can use anyconvenient method, including, e.g., conjugation, bacterialtransformation, calcium-precipitated DNA, electroporation, fusion,transfection, infection with viral vectors, biolistics, and the like.

Accordingly, proteins for use within the present invention can beproduced in genetically engineered host cells according to conventionaltechniques. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells(including cultured cells of multicellular organisms), particularlycultured mammalian cells. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook and Russell, Molecular Cloning: A LaboratoryManual (3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001), and Ausubel et al., Short Protocols in MolecularBiology (4th ed., John Wiley & Sons, 1999).

For example, for recombinant expression of a homodimeric PSMA-bindingprotein comprising two identical PSMA-binding polypeptides as describedherein, an expression vector will generally include a nucleic acidsegment encoding the PSMA-binding polypeptide, operably linked to apromoter. For recombinant expression of a heterodimeric PSMA-bindingprotein, comprising different first and second polypeptide chains, thefirst and second polypeptide chains can be co-expressed from separatevectors in the host cell for expression of the entire heterodimericprotein. Alternatively, for the expression of heterodimeric PSMA-bindingproteins, the first and second polypeptide chains are co-expressed fromseparate expression units in the same vector in the host cell forexpression of the entire heterodimeric protein. The expression vector(s)are transferred to a host cell by conventional techniques, and thetransfected cells are then cultured by conventional techniques toproduce the encoded polypeptide(s) to produce the correspondingPSMA-binding protein.

To direct a recombinant protein into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence) isprovided in the expression vector. The secretory signal sequence can bethat of the native form of the recombinant protein, or can be derivedfrom another secreted protein or synthesized de novo. The secretorysignal sequence is operably linked to the polypeptide-encoding DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences can be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In certainvariations, a secretory signal sequence for use in accordance with thepresent invention has the amino acid sequence MEAPAQLLFLLLLWLPDTTG (SEQID NO:85).

Cultured mammalian cells are suitable hosts for production ofrecombinant proteins for use within the present invention. Methods forintroducing exogenous DNA into mammalian host cells include calciumphosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Vander Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al.,supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed by,for example, Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al.,U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Examples of suitable mammalian hostcells include African green monkey kidney cells (Vero; ATCC CRL 1587),human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamsterkidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), caninekidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1;ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, Utah); see also, e.g.,Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitarycells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells(H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1;ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. Strong transcription promoters can be used, such aspromoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No.4,956,288. Other suitable promoters include those from metallothioneingenes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus majorlate promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants.” Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.”Exemplary selectable markers include a gene encoding resistance to theantibiotic neomycin, which allows selection to be carried out in thepresence of a neomycin-type drug, such as G-418 or the like; the gptgene for xanthine-guanine phosphoribosyl transferase, which permits hostcell growth in the presence of mycophenolic acid/xanthine; and markersthat provide resistance to zeocin, bleomycin, blastocidin, andhygromycin (see, e.g., Gatignol et al., Mol. Gen. Genet. 207:342, 1987;Drocourt et al., Nucl. Acids Res. 18:4009, 1990). Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.An exemplary amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.Transformation of insect cells and production of foreign polypeptidestherein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPOpublication WO 94/06463.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See King and Possee, The Baculovirus Expression System: A LaboratoryGuide (Chapman & Hall, London); O'Reilly et al., Baculovirus ExpressionVectors: A Laboratory Manual (Oxford University Press., New York 1994);and Baculovirus Expression Protocols. Methods in Molecular Biology(Richardson ed., Humana Press, Totowa, N.J., 1995). Recombinantbaculovirus can also be produced through the use of a transposon-basedsystem described by Luckow et al. (J. Virol. 67:4566-4579, 1993). Thissystem, which utilizes transfer vectors, is commercially available inkit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, Md.). Thetransfer vector (e.g., PFASTBAC1; Life Technologies) contains a Tn7transposon to move the DNA encoding the protein of interest into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990;Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk andRapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfervectors can include an in-frame fusion with DNA encoding a polypeptideextension or affinity tag as disclosed above. Using techniques known inthe art, a transfer vector containing a protein-encoding DNA sequence istransformed into E. coli host cells, and the cells are screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, such as Sf9 cells. Recombinant virus thatexpresses the protein or interest is subsequently produced. Recombinantviral stocks are made by methods commonly used in the art.

For protein production, the recombinant virus is used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HIGH FIVEcells; Invitrogen, Carlsbad, Calif.). See generally Glick and Pasternak,Molecular Biotechnology, Principles & Applications of Recombinant DNA(ASM Press, Washington, D.C., 1994). See also U.S. Pat. No. 5,300,435.Serum-free media are used to grow and maintain the cells. Suitable mediaformulations are known in the art and can be obtained from commercialsuppliers. The cells are grown up from an inoculation density ofapproximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells, at which timea recombinant viral stock is added at a multiplicity of infection (MOI)of 0.1 to 10, more typically near 3. Procedures used are generallydescribed in available laboratory manuals (see, e.g., King and Possee,supra; O'Reilly et al., supra; Richardson, supra).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of in this regard include, e.g., Saccharomycescerevisiae, Pichia pastoris, and Pichia methanolica. Methods fortransforming S. cerevisiae cells with exogenous DNA and producingrecombinant polypeptides therefrom are disclosed by, for example,Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No.4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cellsare selected by phenotype determined by the selectable marker, commonlydrug resistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). An exemplary vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Suitable promoters andterminators for use in yeast include those from glycolytic enzyme genes(see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S.Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcoholdehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154;5,139,936; and 4,661,454. Transformation systems for other yeasts,including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichiamethanolica, Pichia guillermondii, and Candida maltose are known in theart. See, e.g., Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986;Cregg, U.S. Pat. No. 4,882,279; and Raymond et al., Yeast 14:11-23,1998. Aspergillus cells can be utilized according to the methods ofMcKnight et al., U.S. Pat. No. 4,935,349. Methods for transformingAcremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No.5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533. Production of recombinant proteinsin Pichia methanolica is disclosed in U.S. Pat. Nos. 5,716,808;5,736,383; 5,854,039; and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus, and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well-known in theart (see, e.g., Sambrook and Russell, supra). When expressing arecombinant protein in bacteria such as E. coli, the protein can beretained in the cytoplasm, typically as insoluble granules, or can bedirected to the periplasmic space by a bacterial secretion sequence. Inthe former case, the cells are lysed, and the granules are recovered anddenatured using, for example, guanidine isothiocyanate or urea. Thedenatured protein can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the alternative, the protein canbe recovered from the cytoplasm in soluble form and isolated without theuse of denaturants. The protein is recovered from the cell as an aqueousextract in, for example, phosphate buffered saline. To capture theprotein of interest, the extract is applied directly to achromatographic medium, such as an immobilized antibody orheparin-Sepharose column. Secreted proteins can be recovered from theperiplasmic space in a soluble and functional form by disrupting thecells (by, for example, sonication or osmotic shock) to release thecontents of the periplasmic space and recovering the protein, therebyobviating the need for denaturation and refolding. Antibodies, includingsingle-chain antibodies, can be produced in bacterial host cellsaccording to known methods. See, e.g., Bird et al., Science 242:423-426,1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; andPantoliano et al., Biochem. 30:10117-10125, 1991.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media can alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

PSMA-binding proteins are purified by conventional protein purificationmethods, typically by a combination of chromatographic techniques. Seegenerally Affinity Chromatography: Principles & Methods (Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification:Principles and Practice (Springer-Verlag, New York 1994). Proteinscomprising an immunoglobulin Fc region can be purified by affinitychromatography on immobilized protein A or protein G. Additionalpurification steps, such as gel filtration, can be used to obtain thedesired level of purity or to provide for desalting, buffer exchange,and the like.

V. Methods of Treatment

In another embodiment, the present invention provides a method fortreating a disorder characterized by overexpression of PSMA. Generally,such methods include administering to a subject in need of suchtreatment a therapeutically effective amount of a PSMA-binding proteinas described herein. In some embodiments, the PSMA-binding proteincomprises at least one effector function selected fromantibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC), such that the PSMA-bindingprotein induces ADCC and/or CDC against PSMA-expressing cells in thesubject. In other embodiments, where the PSMA-binding protein comprisesa second binding domain that specifically binds a T cell (e.g., to a TCRcomplex or component thereof, such as CD3ε), the PSMA-binding proteininduces redirected T-cell cytotoxicity (RTCC) against PSMA-expressingcells in the subject.

In certain variations of the method, the disorder is a cancer. Exemplarycancers amenable to treatment in accordance with the present inventioninclude, for example, prostate cancer (e.g., castrate-resistant prostatecancer), colorectal cancer, gastric cancer, clear cell renal carcinoma,bladder cancer, and lung cancer. In other variations, the disorder is aprostate disorder such as, for example, prostate cancer or benignprostatic hyperplasia (BPH). In yet other embodiments, the disorder isan neovascular disorder such as, for example, a cancer characterized bysolid tumor growth. Exemplary cancers with tumor vasculaturescharacterized by PSMA overexpression and amenable to treatment inaccordance with the present invention include, for example, clear cellrenal carcinoma (CCRCC), colorectal cancer, breast cancer, bladdercancer, lung cancer, and pancreatic cancer (see, e.g., Baccala et al.,Urology 70:385-390, 2007 (expression of PSMA in CCRCC); Liu et al.,Cancer Res. 57:3629-3634, 1997 (expression of PSMA in variousnon-prostate cancers, including renal, urothelial, lung, colon, breast,and adenocarcinaoma to the liver); and Milowsky et al., J. Clin. Oncol.25:540-547, 2007 (expression in, e.g., kidney, colon, bladder, andpancreatic cancers, and demonstration of specific targeting of tumorvasculature in humans using an anti-PSMA mAb).

In each of the embodiments of the treatment methods described herein,the PSMA-binding protein is delivered in a manner consistent withconventional methodologies associated with management of the disease ordisorder for which treatment is sought. In accordance with thedisclosure herein, an effective amount of the PSMA-binding protein isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent or treat the disease or disorder.

Subjects for administration of PSMA-binding proteins as described hereininclude patients at high risk for developing a particular disordercharacterized by PSMA overexpression as well as patients presenting withan existing such disorder. Typically, the subject has been diagnosed ashaving the disorder for which treatment is sought. Further, subjects canbe monitored during the course of treatment for any change in thedisorder (e.g., for an increase or decrease in clinical symptoms of thedisorder). Also, in some variations, the subject does not suffer fromanother disorder requiring treatment that involves targetingPSMA-expressing cells.

In prophylactic applications, pharmaceutical compositions or medicantsare administered to a patient susceptible to, or otherwise at risk of, aparticular disorder in an amount sufficient to eliminate or reduce therisk or delay the onset of the disorder. In therapeutic applications,compositions or medicants are administered to a patient suspected of, oralready suffering from such a disorder in an amount sufficient to cure,or at least partially arrest, the symptoms of the disorder and itscomplications. An amount adequate to accomplish this is referred to as atherapeutically effective dose or amount. In both prophylactic andtherapeutic regimes, agents are usually administered in several dosagesuntil a sufficient response (e.g., inhibition of inappropriateangiogenesis activity) has been achieved. Typically, the response ismonitored and repeated dosages are given if the desired response startsto fade.

To identify subject patients for treatment according to the methods ofthe invention, accepted screening methods can be employed to determinerisk factors associated with specific disorders or to determine thestatus of an existing disorder identified in a subject. Such methods caninclude, for example, determining whether an individual has relativeswho have been diagnosed with a particular disorder. Screening methodscan also include, for example, conventional work-ups to determinefamilial status for a particular disorder known to have a heritablecomponent. For example, various cancers are also known to have certaininheritable components. Inheritable components of cancers include, forexample, mutations in multiple genes that are transforming (e.g., Ras,Raf, EGFR, cMet, and others), the presence or absence of certain HLA andkiller inhibitory receptor (KIR) molecules, or mechanisms by whichcancer cells are able to modulate immune suppression of cells like NKcells and T cells, either directly or indirectly (see, e.g., Ljunggrenand Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann,Clin. Exp. Immunol. 149:1-8, 2007). Toward this end, nucleotide probescan be routinely employed to identify individuals carrying geneticmarkers associated with a particular disorder of interest. In addition,a wide variety of immunological methods are known in the art that areuseful to identify markers for specific disorder. For example, variousELISA immunoassay methods are available and well-known in the art thatemploy monoclonal antibody probes to detect antigens associated withspecific tumors. Screening can be implemented as indicated by knownpatient symptomology, age factors, related risk factors, etc. Thesemethods allow the clinician to routinely select patients in need of themethods described herein for treatment. In accordance with thesemethods, targeting pathological, PSMA-expressing cells can beimplemented as an independent treatment program or as a follow-up,adjunct, or coordinate treatment regimen to other treatments.

For administration, the PSMA-binding protein is formulated as apharmaceutical composition. A pharmaceutical composition comprising aPSMA-binding protein can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby the therapeuticmolecule is combined in a mixture with a pharmaceutically acceptablecarrier. A composition is said to be a “pharmaceutically acceptablecarrier” if its administration can be tolerated by a recipient patient.Sterile phosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well-known to those inthe art. (See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences(Mack Publishing Company, 19th ed. 1995).) Formulations can furtherinclude one or more excipients, preservatives, solubilizers, bufferingagents, albumin to prevent protein loss on vial surfaces, etc.

A pharmaceutical composition comprising a PSMA-binding protein isadministered to a subject in a therapeutically effective amount.According to the methods of the present invention, a PSMA-bindingprotein can be administered to subjects by a variety of administrationmodes, including, for example, by intramuscular, subcutaneous,intravenous, intra-atrial, intra-articular, parenteral, intranasal,intrapulmonary, transdermal, intrapleural, intrathecal, and oral routesof administration. For prevention and treatment purposes, an antagonistcan be administered to a subject in a single bolus delivery, viacontinuous delivery (e.g., continuous transdermal delivery) over anextended time period, or in a repeated administration protocol (e.g., onan hourly, daily, or weekly basis).

A “therapeutically effective amount” of a composition is that amountthat produces a statistically significant effect in amelioration of oneor more symptoms of the disorder, such as a statistically significantreduction in disease progression or a statistically significantimprovement in organ function. The exact dose will be determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.

Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby determining effective dosages and administration protocols thatsignificantly reduce the occurrence or severity of the subject disorderin model subjects. Effective doses of the compositions of the presentinvention vary depending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered,whether treatment is prophylactic or therapeutic, as well as thespecific activity of the composition itself and its ability to elicitthe desired response in the individual. Usually, the patient is a human,but in some diseases, the patient can be a nonhuman mammal. Typically,dosage regimens are adjusted to provide an optimum therapeutic response,i.e., to optimize safety and efficacy. Accordingly, a therapeuticallyeffective amount is also one in which any undesired collateral effectsare outweighed by the beneficial effects of administering a PSMA-bindingprotein as described herein. For administration of the PSMA-bindingprotein, a dosage typically ranges from about 0.1 μg to 100 mg/kg or 1μg/kg to about 50 mg/kg, and more usually 10 μg to 5 mg/kg of thesubject's body weight. In more specific embodiments, an effective amountof the agent is between about 1 μg/kg and about 20 mg/kg, between about10 μg/kg and about 10 mg/kg, or between about 0.1 mg/kg and about 5mg/kg. Dosages within this range can be achieved by single or multipleadministrations, including, e.g., multiple administrations per day ordaily, weekly, bi-weekly, or monthly administrations. For example, incertain variations, a regimen consists of an initial administrationfollowed by multiple, subsequent administrations at weekly or bi-weeklyintervals. Another regimen consists of an initial administrationfollowed by multiple, subsequent administrations at monthly orbi-monthly intervals. Alternatively, administrations can be on anirregular basis as indicated by monitoring clinical symptoms of thedisorder.

Dosage of the pharmaceutical composition can be varied by the attendingclinician to maintain a desired concentration at a target site. Forexample, if an intravenous mode of delivery is selected, localconcentration of the agent in the bloodstream at the target tissue canbe between about 1-50 nanomoles of the composition per liter, sometimesbetween about 1.0 nanomole per liter and 10, 15, or 25 nanomoles perliter depending on the subject's status and projected measured response.Higher or lower concentrations can be selected based on the mode ofdelivery, e.g., trans-epidermal delivery versus delivery to a mucosalsurface. Dosage should also be adjusted based on the release rate of theadministered formulation, e.g., nasal spray versus powder, sustainedrelease oral or injected particles, transdermal formulations, etc. Toachieve the same serum concentration level, for example, slow-releaseparticles with a release rate of 5 nanomolar (under standard conditions)would be administered at about twice the dosage of particles with arelease rate of 10 nanomolar.

Pharmaceutical compositions as described herein can also be used in thecontext of combination therapy. The term “combination therapy” is usedherein to denote that a subject is administered at least onetherapeutically effective dose of a PSMA-binding protein and anothertherapeutic agent.

For example, in the context of cancer immunotherapy, a PSMA-bindingprotein of the present invention can be used in combination withchemotherapy or radiation. A PSMA-binding protein as described hereincan work in synergy with conventional types of chemotherapy orradiation. The PSMA-binding protein can further reduce tumor burden andallow more efficient killing by a chemotherapeutic.

Compositions of the present invention can also be used in combinationwith immunomodulatory compounds including various cytokines andco-stimulatory/inhibitory molecules. These can include, but are notlimited to, the use of cytokines that stimulate anti-cancer immuneresponses (e.g., IL-2, IL-12, or IL-21). In addition, PSMA-bindingproteins can be combined with reagents that co-stimulate various cellsurface molecules found on immune-based effector cells, such as theactivation of CD137 (see Wilcox et al., J. Clin. Invest. 109:651-9,2002) or inhibition of CTLA4 (see Chambers et al., Ann. Rev. Immunol.19:565-94, 2001). Alternatively, PSMA-binding proteins could be usedwith reagents that induce tumor cell apoptosis by interacting with TNFsuperfamily receptors (e.g., TRAIL-related receptors, DR4, DR5, Fas, orCD37). (See, e.g., Takeda et al., J. Exp. Med. 195:161-9, 2002;Srivastava, Neoplasia 3:535-46, 2001.) Such reagents include ligands ofTNF superfamily receptors, including ligand-Ig fusions, and antibodiesspecific for TNF superfamily receptors (e.g., TRAIL ligand, TRAILligand-Ig fusions, anti-TRAIL antibodies, and the like).

With particular regard to treatment of solid tumors, protocols forassessing endpoints and anti-tumor activity are well-known in the art.While each protocol may define tumor response assessments differently,the RECIST (Response evaluation Criteria in solid tumors) criteria iscurrently considered to be the recommended guidelines for assessment oftumor response by the National Cancer Institute (see Therasse et al., J.Natl. Cancer Inst. 92:205-216, 2000). According to the RECIST criteriatumor response means a reduction or elimination of all measurablelesions or metastases. Disease is generally considered measurable if itcomprises lesions that can be accurately measured in at least onedimension as ≧20 mm with conventional techniques or ≧10 mm with spiralCT scan with clearly defined margins by medical photograph or X-ray,computerized axial tomography (CT), magnetic resonance imaging (MRI), orclinical examination (if lesions are superficial). Non-measurabledisease means the disease comprises of lesions <20 mm with conventionaltechniques or <10 mm with spiral CT scan, and truly non-measurablelesions (too small to accurately measure). Non-measureable diseaseincludes pleural effusions, ascites, and disease documented by indirectevidence.

The criteria for objective status are required for protocols to assesssolid tumor response. Representative criteria include the following: (1)Complete Response (CR), defined as complete disappearance of allmeasurable disease; no new lesions; no disease related symptoms; noevidence of non-measurable disease; (2) Partial Response (PR) defined as30% decrease in the sum of the longest diameter of target lesions (3)Progressive Disease (PD), defined as 20% increase in the sum of thelongest diameter of target lesions or appearance of any new lesion; (4)Stable or No Response, defined as not qualifying for CR, PR, orProgressive Disease. (See Therasse et al., supra.)

Additional endpoints that are accepted within the oncology art includeoverall survival (OS), disease-free survival (DFS), objective responserate (ORR), time to progression (TTP), and progression-free survival(PFS) (see Guidance for Industry: Clinical Trial Endpoints for theApproval of Cancer Drugs and Biologics, April 2005, Center for DrugEvaluation and Research, FDA, Rockville, Md.)

Pharmaceutical compositions can be supplied as a kit comprising acontainer that comprises the pharmaceutical composition as describedherein. A pharmaceutical composition can be provided, for example, inthe form of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a pharmaceuticalcomposition. Such a kit can further comprise written information onindications and usage of the pharmaceutical composition.

EXAMPLES Example 1: Isolation of Murine Variable Domains from 107-1A4and Preparation of Humanized Variants

Murine variable domains were cloned from hybridoma cells expressing the107-1A4 monoclonal antibody specific for human PSMA (see Brown et al,1998, Prostate Cancer and Prostatic Diseases. 1: 208-215). Total RNA wasisolated from the hybridoma using RNeasy® Protect Mini kit (QIAGEN Inc.,74124) according to the manufacturer's instructions. SMART™ RACE cDNAamplification kit (Clontech Laboratories, Inc., 634914) was used togenerate 5′RACE-ready cDNA with oligo(dT) primer according to themanufacturer's instructions. V_(H) and V_(L) regions of antibody werePCR-amplified from cDNA by SMART™ RACE protocol using pools ofproprietary degenerate gene specific primers for different murine VK orVH gene families. PCR amplification products were confirmed by gelelectrophoresis, and correct sized bands were isolated and cloned intopCR®2.1-TOPO® plasmid vector using the TOPO® TA Cloning kit according tomanufacturer's instructions (Invitrogen Corporation). The resultingrecombinant vector was transformed into TOP10 E. coli. Sequencing DNAfrom clones revealed multiple isolates of a heavy chain region with amurine VH1 framework with high homology (92.7%) to the murine germlineframework L17134 (GenBank™), and a kappa chain region with a murine Vk16framework with very high homology (98.6%) to the murine germlineframework AJ235936 (EMBL). Two restriction sites—one HindIII and oneEcoRI site—were removed by neutral mutations from the DNA coding for theparent murine kappa (light) variable domain to simplify cloning intodestination mammalian expression vectors, and the native murinesecretion/leader sequences were also not used in favor of the human Vk3leader sequence. The polynucleotide sequence of PSMA-specific murine VHregion (107-1A4) is given in SEQ ID NO:1, and the amino acid sequence isgiven in SEQ ID NO:2. The polynucleotide sequence of PSMA-specificmurine VL region (107-1A4) with the restriction sites is given in SEQ IDNO:3. The polynucleotide sequence of PSMA-specific murine VL region(107-1A4) modified to remove the restriction sites is given in SEQ IDNO:4, and the amino acid sequence is given in SEQ ID NO:5.

DNA sequences coding for these murine scFv sequences and cassetted forinsertion into appropriate scaffolds (e.g., SMIP, SCORPION, andmono-specific or multispecific heterodimer polypeptides) were designed.The constructs were then synthesized by Blue Heron (Bothell, Wash.) andstandard, restriction-digest-based cloning techniques were used toproduce the gene sequences corresponding to TSC084 (SEQ ID NO:44; aminoacid sequence SEQ ID NO:46), TSC085 (SEQ ID NO:36; amino acid sequenceSEQ ID NO:38), and TSC092 (SEQ ID NO:37; amino acid sequence SEQ IDNO:39).

Humanized sequences designed through CDR grafting to human frameworkswere similarly synthesized by Blue Heron and cloned into similar vectorsusing restriction digests to produce the following gene sequences usingtwo approaches: (A) three piece ligation using a HindIII/BamHI fragment,a BamHI/XhoI fragment, and a destination vector cut with HindIII/XhoI toproduce the gene sequences corresponding to TSC188 (SEQ ID NO:40; aminoacid sequence SEQ ID NO:42) and TSC189 (SEQ ID NO:41; amino acidsequence SEQ ID NO:43); and (B) two piece ligation using a HindIII/XhoIfragment and a destination vector cut with HindIII/XhoI to produce thegene sequences corresponding to TSC192 (SEQ ID NO:53; amino acidsequence SEQ ID NO:58), TSC193 (SEQ ID NO:54; amino acid sequence SEQ IDNO:59), TSC194 (SEQ ID NO:48; amino acid sequence SEQ ID NO:49), TSC195(SEQ ID NO:55; amino acid sequence SEQ ID NO:60), TSC196 (SEQ ID NO:56;amino acid sequence SEQ ID NO:61), TSC199 (SEQ ID NO:50; amino acidsequence SEQ ID NO:51), TSC210 (SEQ ID NO:69; amino acid sequence SEQ IDNO:70), TSC211 (SEQ ID NO:71; amino acid sequence SEQ ID NO:72), TSC212(SEQ ID NO:73; amino acid sequence SEQ ID NO:74), TSC213 (SEQ ID NO:75;amino acid sequence SEQ ID NO:76); TSC249 (SEQ ID NO:77; amino acidsequence SEQ ID NO:78), TSC250 (SEQ ID NO:79; amino acid sequence SEQ IDNO:80), TSC251 (SEQ ID NO:81; amino acid sequence SEQ ID NO:82), andTSC252 (SEQ ID NO:83; amino acid sequence SEQ ID NO:84); and (C) twopiece ligation using a BsrGI/EcoRI fragment and one of two destinationvectors cut with BsrGI/EcoRI to produce the gene sequences correspondingto TSC295 (SEQ ID NO:157; amino acid sequence SEQ ID NO:158), TSC296(SEQ ID NO:159; amino acid sequence SEQ ID NO:160), TSC301 (SEQ IDNO:161; amino acid sequence SEQ ID NO:162), and TSC302 (SEQ ID NO:163;amino acid sequence SEQ ID NO:164). The humanized PSMA-specific(107-1A4) VL region polynucleotide sequence is given in SEQ ID NO:22,and the amino acid sequence is given in SEQ ID NO:23. A humanizedPSMA-specific (107-1A4) VH region #1 polynucleotide sequence is given inSEQ ID NO:24, and the amino acid sequence is given in SEQ ID NO:25. Ahumanized PSMA-specific (107-1A4) VH region #2 polynucleotide sequenceis given in SEQ ID NO:26, and the amino acid sequence is given in SEQ IDNO:27.

Sequences for the various cloned sequences and components are alsopresented in Table 3. Amino acid sequences given for polypeptideconstructs (e.g, SMIP, SCORPION, mono- or multi-specific heterodimericproteins) do not include the human Vk3 leader sequence.

TABLE 3 Binding Polypeptide Sequences and Components Amino AcidSEQ ID NOs: Name Nucleotide Sequence Sequence (amino acid) Murine 107-gagatccagctgcaacagtctggacctgagctggtgaagcctggggcttca eiqlqqsgpelvkpgasvkSEQ ID NO: 1 1A4 VHgtgaagatgtcctgcaaggcttctggatacacattcactgactactacatgcac msckasgytftdyymhw(SEQ ID NO: 2) regiontgggtgaagcagaacaatggagagagccttgagtggattggatattttaatcc vkqnngeslewigyfnpyttataatgattatactagatacaaccagaatttcaatggcaaggccacattgactndytrynqnfngkatltvdkgtagacaagtcctccagcacagcctacatgcagctcaacagcctgacatctgssstaymqlnsltsedsafyaggactctgcattctattactgtgcaagatcggatggttactacgatgctatgg ycarsdgyydamdywgqactactggggtcaaggaacctcagtcaccgtctcctcg Murine 107-Gatgtccagataacccagtctccatcttatcttgctgcatctcctggagaaacc SEQ ID NO: 31A4 VL attactattaattgcagggcaagtaagagcattagcaaatatttagcctggtatc region w/aagagaaacctgggaaagctaataagcttcttatccattctggatccactttgc additionalaatctggaattccatcaaggttcagtggcagtggatctggtacagatttcactct restrictioncaccatcagtagcctggagcctgaagattttgcaatgtattactgtcaacagca sitestattgaatacccgtggacgttcggtggtggcaccaaactggaaattaaacgg gct Murine 107-gatgtccagataacccagtctccatcttatcttgctgcatctcctggagaaaccdvqitqspsylaaspgetiti SEQ ID NO: 4 1A4 VLattactattaattgcagggcaagtaagagcattagcaaatatttagcctggtatcncrasksiskylawyciekp (SEQ ID NO: 5) regionaagagaaacctgggaaagctaataagctacttatccattctggatccactttgcgkankllihsgstlqsgipsr modifiedaatctggaataccatcaaggttcagtggcagtggatctggtacagatttcactcfsgsgsgtclftltisslepedftcaccatcagtagcctggagcctgaagattttgcaatgtattactgtcaacagcamyycqqhieypwtfggg atattgaatacccgtggacgttcggtggtggcaccaaactggaaattaaacgtkleikra ggcc 107-1A4 VH tctggatacacattcactgactactacatgcac sgytftdyymhSEQ ID NO: 6 CDR1 (SEQ ID NO: 9) 107-1A4 VHtattttaatccttataatgattatactaga Yfnpyndytr SEQ ID NO: 7 CDR2(SEQ ID NO: 10) 107-1A4 VH tgtgcaagatcggatggttactacgatgctatggactactggcarsdgyydamdyw SEQ ID NO: 8 CDR3 (SEQ ID NO: 11) 107-1A4 VLAagagcattagcaaatat Ksisky SEQ ID NO: 12 CDR1 (SEQ ID NO: 15) 107-1A4 VLTctggatcc Sgs SEQ ID NO: 13 CDR2 (SEQ ID NO: 16) 107-1A4 VLCaacagcatattgaatacccgtggacg Qqhieypwt SEQ ID NO: 14 CDR3 (SEQ ID NO: 17)107-1A4 gagatccagctgcaacagtctggacctgagctggtgaagcctggggcttcaeiqlqqsgpelvkpgasvk SEQ ID NO: 18 VH-VL scFvgtgaagatgtcctgcaaggcttctggatacacattcactgactactacatgcac msckasgytftdyymhw(SEQ ID NO: 19) tgggtgaagcagaacaatggagagagccttgagtggattggatattttaatccvkqnngeslewigyfnpyttataatgattatactagatacaaccagaatttcaatggcaaggccacattgactndynynqnfngkathydk gtagacaagtcctccagcacagcctacatgcagctcaacagcctgacatctgssstaymqlnsltsedsafyaggactctgcattctattactgtgcaagatcggatggttactacgatgctatgg ycarsdgyydamdywgqactactggggtcaaggaacctcagtcaccgtctcctcaggcggcggcggaa gtsvtvssggggsggggssgcggcggtggcggcagcagcggcggcggcggcagcgatgtccagataa ggggsdvqitqspsylaaspcccagtctccatcttatcttgctgcatctcctggagaaaccattactattaattgcgetitincrasksiskylawyagggcaagtaagagcattagcaaatatttagcctggtatcaagagaaacctgqekpgkankllihsgstlqsggaaagctaataagctacttatccattctggatccactttgcaatctggaataccgipsrfsgsgsgtdftltissleatcaaggttcagtggcagtggatctggtacagatttcactctcaccatcagtagpeclfamyycqqhieypwtcctggagcctgaagattttgcaatgtattactgtcaacagcatattgaataccc fgggtkleikrasgtggacgttcggtggtggcaccaaactggaaattaaacgggcctcg 107-1A4gatgtccagataacccagtctccatcttatcttgctgcatctcctggagaaaccdvqitqspsylaaspgetiti SEQ ID NO: 20 VL-VH scFvattactattaattgcagggcaagtaagagcattagcaaatatttagcctggtatcncrasksiskylawyciekp (SEQ ID NO: 21)aagagaaacctgggaaagctaataagctacttatccattctggatccactttgcgkankllihsgstlqsgipsraatctggaataccatcaaggttcagtggcagtggatctggtacagatttcactcfsgsgsgtclftltisslepedftcaccatcagtagcctggagcctgaagattttgcaatgtattactgtcaacagcamyycqqhieypwtfggg atattgaatacccgtggacgttcggtggtggcaccaaactggaaattaaacgtkleikraggggsggggssg ggccggcggcggcggaagcggcggtggcggcagcagcggcggcggcggggseiqlqqsgpelvkpg gcagcgagatccagctgcaacagtctggacctgagctggtgaagcctgggasvkmsckasgytftdyygcttcagtgaagatgtcctgcaaggcttctggatacacattcactgactactac mhwvkqnngeslewigyatgcactgggtgaagcagaacaatggagagagccttgagtggattggatattfnpyndytlynqnfngkatlttaatccttataatgattatactagatacaaccagaatttcaatggcaaggccactvdkssstaymqlnsltsedattgactgtagacaagtcctccagcacagcctacatgcagctcaacagcctg safyycarsdgyydamdyacatctgaggactctgcattctattactgtgcaagatcggatggttactacgatg wgqgtsvtvssctatggactactggggtcaaggaacctcagtcaccgtctcctcg Humanizedgatatccagatgacccagtctccatccgccatgtctgcatctgtaggagacag diqmtqspsamsasvgdrSEQ ID NO: 22 107-1A4 VLagtcaccatcacttgccgggcgagtaagagcattagcaaatatttagcctggtvtitcrasksiskylawfqqk (SEQ ID NO: 23)ttcagcagaaaccagggaaagttcctaagctccgcatccattctggatctacttpgkvpktrihsgstlqsgyptgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagaatttsrfsgsgsgteftltisslqpeactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaadfatyycqqhieypivtfgq cagcatattgaatacccgtggacgttcggccaagggaccaaggtggaaatcgtkveikr aaacga Humanizedgaggtccagctggtacagtctggggctgaggtgaagaagcctggggctac evqlvqsgaeykkpgatvkSEQ ID NO: 24 107-1A4agtgaagatctcctgcaaggcttctggatacacattcactgactactacatgcaisckasgytftdyymhwv (SEQ ID NO: 25) VH#1ctgggtgcaacaggcccctggaaaagggcttgagtggatgggatattttaat qqapgkglewmgyfnpyccttataatgattatactagatacgcagagaagttccagggcagagtcaccatndytiyaekfqgivtitadts aaccgcggacacgtctacagacacagcctacatggagctgagcagcctgatdtaymelsslrsedtavyygatctgaggacacggccgtgtattactgtgcaagatcggatggttactacgat carsdgyydamdywgqggctatggactactggggtcaaggaaccacagtcaccgtctcctcg ttytyss Humanizedcaggtccagctggtacagtctggggctgaggtgaagaagcctggggcttca qvqlvqsgaeykkpgasySEQ ID NO: 26 107-1A4gtgaaggtctcctgcaaggcttctggatacacattcactgactactacatgcackvsckasgytftdyymhw (SEQ ID NO: 27) VH#2tgggtgcgacaggcccctggacaagggcttgagtggatgggatattttaatc vrqapgqglewmgyfnpcttataatgattatactagatacgcacagaagttccagggcagagtcaccatgyndytiyaqkfqgrvtmtr accagggacacgtctatcagcacagcctacatggagctgagcagcctgagadtsistaymelsslrsddtavtctgacgacacggccgtgtattactgtgcaagatcggatggttactacgatgct yycarsdgyydamdywgatggactactggggtcaaggaaccacagtcaccgtctcctcg qgttytyss Humanizedgatatccagatgacccagtctccatccgccatgtctgcatctgtaggagacag diqmtqspsamsasvgdrSEQ ID NO: 28 107-1A4agtcaccatcacttgccgggcgagtaagagcattagcaaatatttagcctggtvtitcrasksiskylawfqqk (SEQ ID NO: 30) VL-VH#1ttcagcagaaaccagggaaagttcctaagctccgcatccattctggatctacttpgkvpklrihsgstlqsgvp scFvtgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagaatttsrfsgsgsgteftltisslqpeactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaadfatyycqqhieypwtfgq cagcatattgaatacccgtggacgttcggccaagggaccaaggtggaaatcgtkveikrggggsggggsg aaacgaggtggcggagggtctgggggtggcggatccggaggtggtggctgggsevqlvqsgaevkkp ctgaggtccagctggtacagtctggggctgaggtgaagaagcctggggctagatvkisckasgytftdyycagtgaagatctcctgcaaggcttctggatacacattcactgactactacatgc mhwvqqapgkglewmgactgggtgcaacaggcccctggaaaagggcttgagtggatgggatattttaayfnpyndytiyaekfqgrvttccttataatgattatactagatacgcagagaagttccagggcagagtcaccatitadtstdtaymelsslrsedtaaccgcggacacgtctacagacacagcctacatggagctgagcagcctga avyycarsdgyydamdygatctgaggacacggccgtgtattactgtgcaagatcggatggttactacgat wgqgttvtvssgctatggactactggggtcaaggaaccacagtcaccgtctcctcg Humanizedgatatccagatgacccagtctccatccgccatgtctgcatctgtaggagacag diqmtqspsamsasvgdrSEQ ID NO: 29 107-1A4agtcaccatcacttgccgggcgagtaagagcattagcaaatatttagcctggtvtitcrasksiskylawfqqk (SEQ ID NO: 31) VL-VH#2ttcagcagaaaccagggaaagttcctaagctccgcatccattctggatctacttpgkvpklrihsgstlqsgvp scFvtgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagaatttsrfsgsgsgteftltisslqpeactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaadfatyycqqhieypwtfgq cagcatattgaatacccgtggacgttcggccaagggaccaaggtggaaatcgtkveikrggggsggggsg aaacgaggtggcggagggtctgggggtggcggatccggaggtggtggctgggsqvqlvqsgaevkkp ctcaggtccagctggtacagtctggggctgaggtgaagaagcctggggcttgasvkvsckasgytftdyycagtgaaggtctcctgcaaggcttctggatacacattcactgactactacatgc mhwvrqapgqglewmgactgggtgcgacaggcccctggacaagggcttgagtggatgggatattttaa yfnpyndytiyaqkfqgrytccttataatgattatactagatacgcacagaagttccagggcagagtcaccattmtrdtsistaymelsslrsdgaccagggacacgtctatcagcacagcctacatggagctgagcagcctgag dtavyycarsdgyydamdatctgacgacacggccgtgtattactgtgcaagatcggatggttactacgatg ywgqgttvtvssctatggactactggggtcaaggaaccacagtcaccgtctcctcg Humanizedgaggtccagctggtacagtctggggctgaggtgaagaagcctggggctac evqlvqsgaevkkpgatvkSEQ ID NO: 32 107-1A4agtgaagatctcctgcaaggcttctggatacacattcactgactactacatgcaisckasgytftdyymhwv (SEQ ID NO: 34) VH#1-VLctgggtgcaacaggcccctggaaaagggcttgagtggatgggatattttaat qqapgkglewmgyfnpyscFv ccttataatgattatactagatacgcagagaagttccagggcagagtcaccatndytiyaekfqgivtitadts aaccgcggacacgtctacagacacagcctacatggagctgagcagcctgatdtaymelsslrsedtavyygatctgaggacacggccgtgtattactgtgcaagatcggatggttactacgat carsdgyydamdywgqggctatggactactggggtcaaggaaccacagtcaccgtctcctcaggtggcg ttvtvssggggsggggsgggagggtctgggggtggcggatccggaggtggtggctctgatatccagatga ggsdiqmtqspsamsasvcccagtctccatccgccatgtctgcatctgtaggagacagagtcaccatcactgdivtitcrasksiskylawftgccgggcgagtaagagcattagcaaatatttagcctggtttcagcagaaaccqqkpgkvpklrihsgstlqsagggaaagttcctaagctccgcatccattctggatctactttgcaatcaggggtgvpsrfsgsgsgteftltisslcccatctcggttcagtggcagtggatctgggacagaatttactctcaccatcaqpalfatyycqqhieypwtgcagcctgcagcctgaagattttgcaacttattactgtcaacagcatattgaata fgqgtkveikrascccgtggacgttcggccaagggaccaaggtggaaatcaaacgagcctcg Humanizedcaggtccagctggtacagtctggggctgaggtgaagaagcctggggcttca qvqlvqsgaevkkpgasvSEQ ID NO: 33 107-1A4gtgaaggtctcctgcaaggcttctggatacacattcactgactactacatgcackvsckasgytftdyymhw (SEQ ID NO: 35) VH#2-VLtgggtgcgacaggcccctggacaagggcttgagtggatgggatattttaatc vrqapgqglewmgyfnpscFv cttataatgattatactagatacgcacagaagttccagggcagagtcaccatgyndytiyaqkfqgrvtmtr accagggacacgtctatcagcacagcctacatggagctgagcagcctgagadtsistaymelsslrsddtavtctgacgacacggccgtgtattactgtgcaagatcggatggttactacgatgct yycarsdgyydamdywgatggactactggggtcaaggaaccacagtcaccgtctcctcaggtggcgga qgttvtvssggggsggggsgggtctgggggtggcggatccggaggtggtggctctgatatccagatgacc ggggsdiqmtqspsamsacagtctccatccgccatgtctgcatctgtaggagacagagtcaccatcacttgsvgdivtitcrasksiskylaccgggcgagtaagagcattagcaaatatttagcctggtttcagcagaaaccawfqqkpgkvpklrihsgstlgggaaagttcctaagctccgcatccattctggatctactttgcaatcaggggtcqsgvpsrfsgsgsgteftltisccatctcggttcagtggcagtggatctgggacagaatttactctcaccatcagslqpedfatyycqqhieypcagcctgcagcctgaagattttgcaacttattactgtcaacagcatattgaatac wtfgqgtkveikrasccgtggacgttcggccaagggaccaaggtggaaatcaaacgcgcctcg TSC085atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatacdvqitqspsylaaspgetiti SEQ ID NO: 36 chimericcaccggtgatgtccagataacccagtctccatcttatcttgctgcatctcctggancrasksiskylawyqekp (SEQ ID NO: 38) SMIPgaaaccattactattaattgcagggcaagtaagagcattagcaaatatttagccgkankllihsgstlqsgipsr (murine 107-tggtatcaagagaaacctgggaaagctaataagctacttatccattctggatccfsgsgsgtclftltisslepedf 1A4 VL-VHactttgcaatctggaataccatcaaggttcagtggcagtggatctggtacagatamyycqqhieypwtfggg scFv-humanttcactctcaccatcagtagcctggagcctgaagattttgcaatgtattactgtctkleikraggggsggggssg Fc)aacagcatattgaatacccgtggacgttcggtggtggcaccaaactggaaattgggseiqlqqsgpelvkpg aaacgggccggcggcggcggaagcggcggtggcggcagcagcggcggasvkmsckasgytftdyy cggcggcagcgagatccagctgcaacagtctggacctgagctggtgaagcmhwvkqnngeslewigy ctggggcttcagtgaagatgtcctgcaaggcttctggatacacattcactgactfnpyndytiynqnfngkatl actacatgcactgggtgaagcagaacaatggagagagccttgagtggattgtvdkssstaymqlnsltsedgatattttaatccttataatgattatactagatacaaccagaatttcaatggcaagsafyycarsdgyydamdy gccacattgactgtagacaagtcctccagcacagcctacatgcagctcaacawgqgtsvtvsssepkssdkgcctgacatctgaggactctgcattctattactgtgcaagatcggatggttactathtcppcpapeaagapsvflcgatgctatggactactggggtcaaggaacctcagtcaccgtctcctcgagtfppkpkdtlmisrtpevtcvgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctg vvdvshedpevkfnwyvaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacac dgvevhnaktkpreeqynscctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagctyrvvsyltvlhqdwhigka cacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgyacaysnkalpapiektisk cataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgakgqprepqvytlppsrdel tgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggctknqvsltclvkgfypsdia gtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaaacvewesngqpennykttpp catctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcvldsdgsfflyskltvdksr ccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggwqqgnvfscsvmhealhn tcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggchytqkslslspgk agccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga TSC092atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataceiqlqqsgpelvkpgasvk SEQ ID NO: 37 chimericcaccggtgagatccagctgcaacagtctggacctgagctggtgaagcctgg msckasgytftdyymhw(SEQ ID NO: 39) SMIPggcttcagtgaagatgtcctgcaaggcttctggatacacattcactgactactavkqnngeslewigyfnpy (murine 107-catgcactgggtgaagcagaacaatggagagagccttgagtggattggatatndytiynqnfngkatltvdk 1A4 VH-VLtttaatccttataatgattatactagatacaaccagaatttcaatggcaaggccassstaymqlnsltsedsafy scFv-humancattgactgtagacaagtcctccagcacagcctacatgcagctcaacagcct ycarsdgyydamdywgqFc) gacatctgaggactctgcattctattactgtgcaagatcggatggttactacgatgtsvtvssggggsggggss gctatggactactggggtcaaggaacctcagtcaccgtctcctcaggcggcgggggsdvqitqspsylaasp gcggaagcggcggtggcggcagcagcggcggcggcggcagcgatgtccgetitincrasksiskylawyagataacccagtctccatcttatcttgctgcatctcctggagaaaccattactattqekpgkankllihsgstlqsaattgcagggcaagtaagagcattagcaaatatttagcctggtatcaagagaagipsrfsgsgsgtdftltissleacctgggaaagctaataagctacttatccattctggatccactttgcaatctggapeclfamyycqqhieypwtataccatcaaggttcagtggcagtggatctggtacagatttcactctcaccatcfgggtkleikrassepkssdagtagcctggagcctgaagattttgcaatgtattactgtcaacagcatattgaatkthtcppcpapeaagapsv acccgtggacgttcggtggtggcaccaaactggaaattaaacgggcctcgaflfppkpkdtlmisrtpevtcgtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacc vvvdvshedpevkfnwytgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggac vdgvevhnaktkpreeqyaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtganstyrvvsyltvlhqdwhig gccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgk gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga TSC188atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 40 humanizedcaccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 42) SMIP (107-gagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp 1A4 VL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#1 scFv-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq Fc)acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggtgggsevqlvqsgaevkkp ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgatvkisckasgytftdyy ggtggctctgaggtccagctggtacagtctggggctgaggtgaagaagcctmhwvqqapgkglewmg ggggctacagtgaagatctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaekfqgrvt ctacatgcactgggtgcaacaggcccctggaaaagggcttgagtggatgggitadtstdtaymelsslrsedtatattttaatccttataatgattatactagatacgcagagaagttccagggcagaavyycarsdgyydamdy gtcaccataaccgcggacacgtctacagacacagcctacatggagctgagcwgqgttvtvsssepkssdktagcctgagatctgaggacacggccgtgtattactgtgcaagatcggatggtthtcppcpapeaagapsvflfactacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcppkpkdtlmisrtpevtcvvgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca vdvshedpevkfnwyvdcctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagg gvevhnaktkpreeqynstacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgt yrvvsyltvlhqdwhigkagagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga yacaysnkalpapiektiskggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt akgqprepqvytlppsrdelaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaatknqvsltclvkgfypsdia ggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagavewesngqpennykttpp aaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccvldsdgsfflyskltvdksr ctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcwqqgnvfscsvmhealhn ctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaathytqkslslspgk gggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga TSC189atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 41 humanizedcaccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 43) SMIP (107-gagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp 1A4 VL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 scFv-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq Fc)acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggtgggsqvqlvqsgaevkkp ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcctmhwvrqapgqglewmg ggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgk gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga TSC084atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatacdvqitqspsylaaspgetiti SEQ ID NO: 44 chimericcaccggtgatgtccagataacccagtctccatcttatcttgctgcatctcctggancrasksiskylawyqekp (SEQ ID NO: 46) Interceptorgaaaccattactattaattgcagggcaagtaagagcattagcaaatatttagccgkankllihsgstlqsgipsr (murine VL-tggtatcaagagaaacctgggaaagctaataagctacttatccattctggatccfsgsgsgtclftltisslepedf VH 107-1A4actttgcaatctggaataccatcaaggttcagtggcagtggatctggtacagatamyycqqhieypwtfggg scFv-Fc-ttcactctcaccatcagtagcctggagcctgaagattttgcaatgtattactgtctkleikraggggsggggssg CH1)aacagcatattgaatacccgtggacgttcggtggtggcaccaaactggaaattgggseiqlqqsgpelvkpg aaacgggccggcggcggcggaagcggcggtggcggcagcagcggcggasvkmsckasgytftdyy cggcggcagcgagatccagctgcaacagtctggacctgagctggtgaagcmhwvkqnngeslewigy ctggggcttcagtgaagatgtcctgcaaggcttctggatacacattcactgactfnpyndytiynqnfngkatl actacatgcactgggtgaagcagaacaatggagagagccttgagtggattgtvdkssstaymqlnsltsedgatattttaatccttataatgattatactagatacaaccagaatttcaatggcaagsafyycarsdgyydamdy gccacattgactgtagacaagtcctccagcacagcctacatgcagctcaacawgqgtsvtvsssepkssdkgcctgacatctgaggactctgcattctattactgtgcaagatcggatggttactathtcppcpapeaagapsvflcgatgctatggactactggggtcaaggaacctcagtcaccgtctcctcgagcfppkpkdtlmisrtpevtcvgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctg vvdvshedpevkfnwyvaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacac dgvevhnaktkpreeqynscctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagctyrvvsyltvlhqdwhigka cacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgyacaysnkalpapiektisk cataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgakgqprepqvytlppsrdel tgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggctknqvsltclvkgfypsdia gtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaaacvewesngqpennykUpp catctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcvldsdgsfflyskltvdksr ccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggwqqgnvfscsvmhealhn tcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggchytqkslslspgksrastkg agccggagaacaactacaagaccacgcctcccgtgctggactccgacggcpsvfplapsskstsggtaalgtccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagg clvkdyfpepvtvswnsgaggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgltsgvhtfpavlqssglyslsscagaagagcctctccctgtctccgggtaaatctagagcctccaccaagggccvvtvpssslgtqtyicnvnhcatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagc kpsntkvdkkvggccctgggctgcctggtcaaggactacttccccgagccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtttga TSC093atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatacqvqlvqsgggvvqpgrslrl SEQ ID NO: 45 Interceptorcaccggtcaggtccagctggtgcagtctgggggcggagtggtgcagcctg sckasgytftrstmhwvrq(SEQ ID NO: 47) (Cris7 scFv-ggcggtcactgaggctgtcctgcaaggcttctggctacacctttactagatctaapgkglewigyinpssayt Fc-Cκ_(YAE))cgatgcactgggtaaggcaggcccctggaaagggtctggaatggattggat nynqkfkdrftisadkskstacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttaflqmdslipedtgvyfcar cacaatcagcgcagacaaatccaagagcacagccttcctgcagatggacagpqvhydyngfpywgqgt cctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccapvtvssggggsggggsggctatgattacaacgggtttccttactggggccaagggactcccgtcactgtctcggsaqdiqmtqspsslsas tagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgvgdrvtmtcsasssysymn cacaagacatccagatgacccagtctccaagcagcctgtctgcaagcgtggwyqqkpgkapkrwiydss gggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatgaaklasgvparfsgsgsgtdytlctggtaccagcagaagcccggcaaggcccccaaaagatggatttatgactc tisslqpedfatyycqqwsratccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctggganpptfgggtklqitrssepksccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttatsdkthtcppcpapeaagap tactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaasvflfppkpkdtlmisrtpegctacaaattacacgctcgagtgagcccaaatcttctgacaaaactcacacat vtcvvvdvshedpevkfngcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctctt wyvdgvevhnaktkpreeccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcac qynstyrvvsyltvlhqdwlatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactg ngkayacaysnkalpapiegtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggag ktiskakgqprepqvytlppgagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccsrdeltknqvsltclvkgfyp aggactggctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagccsdiavewesngqpennyk ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgttppvldsdgsfflyskltvdagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa ksrwqqgnvfscsvmheaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgcclhnhytqkslslspgksrtva gtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcapsvfifppsdeqlksgtasctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtgvvcllnyfypreakvqwkv gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatdnalqsgnsqesateqdsk gaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtadstyslsseltlskadyekhkaatctagaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcavyacevthqglsspvtksfngttgaaatctggaactgcctctgttgtgtgcctgctgaattacttctatcccaga rgegaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgccacagagcaggacagcaaggacagcacctacagcctcagcagcgagctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttc aacaggggagagtgaTSC194 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatacdiqmtqspsamsasvgdr SEQ ID NO: 48 Scorpioncaccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 49) (huVL-gagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp VH#2 107-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe 1A4 scFv-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq Fc-Cris7acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg scFv)actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggt gasvkvsckasgytftdyyggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvrqapgqglewmgggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgqrhnns gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgslntgtqmaghspnsqvq1 gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcavqsgggvvqpgrslrlsckaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacasgytftrstmhwvrqapgk cgcagaagagcctctccctgtctccgggtcagaggcacaacaattcttccctglewigyinpssaytnynqgaatacaggaactcagatggcaggtcattctccgaattctcaggtccagctggkfkdrftisadkskstaflqmtgcagtctgggggcggagtggtgcagcctgggcggtcactgaggctgtcct dslrpedtgvyfcarpqvhygcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcag dyngfpywgqgtpvtvssgcccctggaaagggtctggaatggattggatacattaatcctagcagtgcttatggggsggggsggggsaqd actaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatciqmtqspsslsasvgdrvt caagagcacagccttcctgcagatggacagcctgaggcccgaggacaccgmtcsasssysymnwyqqgcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttakpgkapkrwiydssklasg ctggggccaagggactcccgtcactgtctctagcggtggcggagggtctggvparfsgsgsgtdytltisslqgggtggcggatccggaggtggtggctctgcacaagacatccagatgaccc pedfatyycqqwsmpptfagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacct gggtklqitrgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagcccggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacacgataa TSC199atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 50 Scorpioncaccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 51) (huVL-gagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp VH#1 107-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe 1A4 scFv-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq Fc-Cris7acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg scFv)actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsevqlvqsgaevkkpggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggt gatvkisckasgytftdyyggtggctctgaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvqqapgkglewmgggggctacagtgaagatctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaekfqgrvt ctacatgcactgggtgcaacaggcccctggaaaagggcttgagtggatgggitadtstdtaymelsslrsedtatattttaatccttataatgattatactagatacgcagagaagttccagggcagaavyycarsdgyydamdy gtcaccataaccgcggacacgtctacagacacagcctacatggagctgagcwgqgttvtvsssepkssdktagcctgagatctgaggacacggccgtgtattactgtgcaagatcggatggtthtcppcpapeaagapsvflfactacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcppkpkdtlmisrtpevtcvvgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca vdvshedpevkfnwyvdcctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagg gvevhnaktkpreeqynstacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgt yrvvsyltvlhqdwhigkagagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga yacaysnkalpapiektiskggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt akgqprepqvytlppsrdelaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaatknqvsltclvkgfypsdia ggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagavewesngqpennykUpp aaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccvldsdgsfflyskltvdksr ctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcwqqgnvfscsvmhealhn ctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaathytqkslslspgqrhnnssl gggcagccggagaacaactacaagaccacgcctcccgtgctggactccgantgtqmaghspnsqvqlv cggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagqsgggvvqpgrslrlsckascaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactagytftrstmhwvrqapgkg cacgcagaagagcctctccctgtctccgggtcagaggcacaacaattcttcclewigyinpssaytnynqkctgaatacaggaactcagatggcaggtcattctccgaattctcaggtccagctfkdrftisadkskstaflqmd ggtgcagtctgggggcggagtggtgcagcctgggcggtcactgaggctgtslrpedtgvyfcarpqvhydcctgcaaggcttctggctacacctttactagatctacgatgcactgggtaagg yngfpywgqgtpvtvssgcaggcccctggaaagggtctggaatggattggatacattaatcctagcagtg gggsggggsggggsaqdicttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcagacqmtqspsslsasvgdrvtm aaatccaagagcacagccttcctgcagatggacagcctgaggcccgaggatcsasssysymnwyqqkpcaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgggttgkapkrwiydssklasgvp tccttactggggccaagggactcccgtcactgtctctagcggtggcggaggarfsgsgsgtdytltisslqpegtctgggggtggcggatccggaggtggtggctctgcacaagacatccagat dfatyycqqwsrnpptfgggacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccat gtklqitrgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagcccggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacacga taa TSC125atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatacqvqlvqsgggvvqpgrslrl SEQ ID NO: 52 Interceptorcaccggtcaggtccagctggtgcagtctgggggcggagtggtgcagcctg sckasgytftrstmhwvrq(SEQ ID NO: 57) (Cris7 scFv-ggcggtcactgaggctgtcctgcaaggcttctggctacacctttactagatctaapgkglewigyinpssayt Fc-CH1)cgatgcactgggtaaggcaggcccctggaaagggtctggaatggattggat nynqkfkdrftisadkskstacattaatcctagcagtgcttatactaattacaatcagaaattcaaggacaggttaflqmdshpedtgvyfcar cacaatcagcgcagacaaatccaagagcacagccttcctgcagatggacagpqvhydyngfpywgqgt cctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagtccapvtvssggggsggggsggctatgattacaacgggtttccttactggggccaagggactcccgtcactgtctcggsaqdiqmtqspsslsas tagcggtggcggagggtctgggggtggcggatccggaggtggtggctctgvgdrvtmtcsasssysymn cacaagacatccagatgacccagtctccaagcagcctgtctgcaagcgtggwyqqkpgkapkrwiydss gggacagggtcaccatgacctgcagtgccagctcaagtgtaagttacatgaaklasgvparfsgsgsgtdytlctggtaccagcagaagcccggcaaggcccccaaaagatggatttatgactc tisslqpedfatyycqqwsratccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctggganpptfgggtklqitrssepksccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttatsdkthtcppcpapeaagap tactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaasvflfppkpkdtlmisrtpegctacaaattacacgctcgagtgagcccaaatcttctgacaaaactcacacat vtcvvvdvshedpevkfngcccaccgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctctt wyvdgvevhnaktkpreeccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcac qynstyrvvsyltvlhqdwlatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactg ngkayacaysnkalpapiegtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggag ktiskakgqprepqvytlppgagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccsrdeltknqvsltclvkgfyp aggactggctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagccsdiavewesngqpennyk ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgttppvldsdgsfflyskltvdagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa ksrwqqgnvfscsvmheaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgcclhnhytqkslslspgksrast gtggagtgggagagcaatgggcagccggagaacaactacaagaccacgckgpsvfplapsskstsggtactcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtgalgclvkdyfpepvtvswn gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatsgaltsgvhtfpavlqssglygaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaslssvvtvpssslgtqtyicnaatctagagcctccaccaagggcccatcggtcttccccctggcaccctcctc vnlikpsntkvdavcaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgagccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtttga TSC192atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 53 Interceptorcaccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 58) (huVL-gagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp VH#2 107-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe 1A4 scFv-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq Fc-Cκ_(YAE))acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggtgggsqvqlvqsgaevkkp ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcctmhwvrqapgqglewmg ggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytryaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgksrtva gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgapsvfifppsdeqlksgtasgctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca vvanyfypreakvqwkyggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacadnalqsgnsqesateqdsk cgcagaagagcctctccctgtctccgggtaaatctagaactgtggctgcaccdstyslsseltlskadyekhkatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctvyacevthqglsspvtksfngttgtgtgcctgctgaattacttctatcccagagaggccaaagtacagtggaa rgeggtggataacgccctccaatcgggtaactcccaggagagtgccacagagcaggacagcaaggacagcacctacagcctcagcagcgagctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtga TSC193atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 54 Interceptorcaccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 59) (huVL-gagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp VH#1 107-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe 1A4 scFv-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq Fc-Cκ_(YAE))acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggtgggsevqlvqsgaevkkp ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgatvkisckasgytftdyy ggtggctctgaggtccagctggtacagtctggggctgaggtgaagaagcctmhwvqqapgkglewmg ggggctacagtgaagatctcctgcaaggcttctggatacacattcactgactayfnpyndytryaekfqgrvt ctacatgcactgggtgcaacaggcccctggaaaagggcttgagtggatgggitadtstdtaymelsslrsedtatattttaatccttataatgattatactagatacgcagagaagttccagggcagaavyycarsdgyydamdy gtcaccataaccgcggacacgtctacagacacagcctacatggagctgagcwgqgttvtvsssepkssdktagcctgagatctgaggacacggccgtgtattactgtgcaagatcggatggtthtcppcpapeaagapsvflfactacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcppkpkdtlmisrtpevtcvvgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca vdvshedpevkfnwyvdcctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagg gvevhnaktkpreeqynstacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgt yrvvsyltvlhqdwhigkagagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga yacaysnkalpapiektiskggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt akgqprepqvytlppsrdelaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaatknqvsltclvkgfypsdia ggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagavewesngqpennykttpp aaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccvldsdgsfflyskltvdksr ctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcwqqgnvfscsvmhealhn ctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaathytqkslslspgksrtvaaps gggcagccggagaacaactacaagaccacgcctcccgtgctggactccgavfifppsdeqlksgtasvvclcggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcag lnyfypreakvqwkvdnalcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaqsgnsqesateqdskdstyscacgcagaagagcctctccctgtctccgggtaaatctagaactgtggctgcalsseltlskadyekhkvyacccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctevthqglsspvtksfnrgectgttgtgtgcctgctgaattacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgccacagagcaggacagcaaggacagcacctacagcctcagcagcgagctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtga TSC195atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 55 Interceptorcaccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 60) (huVL-gagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp VH#2 107-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe 1A4 scFv-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq Fc-CH1)acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggtgggsqvqlvqsgaevkkp ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcctmhwvrqapgqglewmg ggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytryaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgksrast gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgkgpsvfplapsskstsggtagctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca algclvkdyfpepvtvswnggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacasgaltsgvhtfpavlqssglycgcagaagagcctctccctgtctccgggtaaatctagagcctccaccaagggslssvvtvpssslgtqtyicncccatcggtcttccccctggcaccctcctccaagagcacctctgggggcaca vnlikpsntkvdavgcggccctgggctgcctggtcaaggactacttccccgagccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtttga TSC196atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 56 Interceptorcaccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 61) (huVL-gagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp VH#1 107-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe 1A4 scFv-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq Fc-CH1)acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggtgggsevqlvqsgaevkkp ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgatvkisckasgytftdyy ggtggctctgaggtccagctggtacagtctggggctgaggtgaagaagcctmhwvqqapgkglewmg ggggctacagtgaagatctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaekfqgrvt ctacatgcactgggtgcaacaggcccctggaaaagggcttgagtggatgggitadtstdtaymelsslrsedtatattttaatccttataatgattatactagatacgcagagaagttccagggcagaavyycarsdgyydamdy gtcaccataaccgcggacacgtctacagacacagcctacatggagctgagcwgqgttvtvsssepkssdktagcctgagatctgaggacacggccgtgtattactgtgcaagatcggatggtthtcppcpapeaagapsvflfactacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcppkpkdtlmisrtpevtcvvgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca vdvshedpevkfnwyvdcctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagg gvevhnaktkpreeqynstacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgt yrvvsyltvlhqdwhigkagagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga yacaysnkalpapiektiskggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt akgqprepqvytlppsrdelaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaatknqvsltclvkgfypsdia ggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagavewesngqpennykttpp aaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccvldsdgsfflyskltvdksr ctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcwqqgnvfscsvmhealhn ctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaathytqkslslspgksrastkg gggcagccggagaacaactacaagaccacgcctcccgtgctggactccgapsvfplapsskstsggtaalgcggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcag clvkdyfpepvtvswnsgacaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaltsgvhtfpavlqssglyslsscacgcagaagagcctctccctgtctccgggtaaatctagagcctccaccaagvvtvpssslgtqtyicnvnhggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggca kpsntkvdkkvcagcggccctgggctgcctggtcaaggactacttccccgagccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtttga TSC210atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac qvqlvqsgaeykkpgasySEQ ID NO: 69 humanizedcaccggtcaggtccagctggtacagtctggggctgaggtgaagaagcctgg kvsckasgytftdyymhw(SEQ ID NO: 70) SMIPggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactacta vrqapgqglewmgyfnp(human catgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatayndytiyaqkfqgrvtmtr VH#2-VLttttaatccttataatgattatactagatacgcacagaagttccagggcagagtcdtsistaymelsslrsddtav scFv-Fc)accatgaccagggacacgtctatcagcacagcctacatggagctgagcagc yycarsdgyydamdywgctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttactaqgttvtvssggggsggggs cgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcaggtggggsdiqmtqspsamsa ggcggagggtctgggggtggcggatccggaggtggtggctctgatatccasvgdivtitcrasksiskylagatgacccagtctccatccgccatgtctgcatctgtaggagacagagtcaccwfqqkpgkvpklrihsgstlatcacttgccgggcgagtaagagcattagcaaatatttagcctggtttcagcaqsgvpsrfsgsgsgteftltisgaaaccagggaaagttcctaagctccgcatccattctggatctactttgcaatcslqpedfatyycqqhieypaggggtcccatctcggttcagtggcagtggatctgggacagaatttactctcawtfgqgtkveikrassepksccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcatasdkthtcppcpapeaagap ttgaatacccgtggacgttcggccaagggaccaaggtggaaatcaaacgcgsvflfppkpkdtomisrtpecctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgccc vtcyvvdvshedpevkfnagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccc wyydgvevhnaktkpreeaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggqynstyrvvsyltylhqdwl acgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgngkayacaysnkalpapie tggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcktiskakgqprepqvytlppacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgsrdeltknqvsltclykgfyp gcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcsdiavewesngqpennyk gagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtattppyldsdgsfflyskltydcaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac ksrwqqgnvfscsvmheactgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagag lhnhytqkslslspgkcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga TSC211atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatacevqlvqsgaeykkpgatvk SEQ ID NO: 71 humanizedcaccggtgaggtccagctggtacagtctggggctgaggtgaagaagcctg isckasgytftdyymhwv(SEQ ID NO: 72) SMIPgggctacagtgaagatctcctgcaaggcttctggatacacattcactgactact qqapgkglewmgyfnpy(human acatgcactgggtgcaacaggcccctggaaaagggcttgagtggatgggatndytiyaekfqgivtitadts VH#1 -VLattttaatccttataatgattatactagatacgcagagaagttccagggcagagttdtaymelsslrsedtavyy scFv-Fc)caccataaccgcggacacgtctacagacacagcctacatggagctgagcag carsdgyydamdywgqgcctgagatctgaggacacggccgtgtattactgtgcaagatcggatggttactttytyssggggsggggsgg acgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcaggggsdiqmtqspsamsasv tggcggagggtctgggggtggcggatccggaggtggtggctctgatatccagdivtitcrasksiskylawfgatgacccagtctccatccgccatgtctgcatctgtaggagacagagtcaccqqkpgkvpklrihsgstlqsatcacttgccgggcgagtaagagcattagcaaatatttagcctggtttcagcagypsrfsgsgsgteftltisslgaaaccagggaaagttcctaagctccgcatccattctggatctactttgcaatcqpalfatyycqqhieypwtaggggtcccatctcggttcagtggcagtggatctgggacagaatttactctcafgqgtkveikrassepkssdccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcatakthtcppcpapeaagapsv ttgaatacccgtggacgttcggccaagggaccaaggtggaaatcaaacgagflfppkpkdtlmisrtpevtccctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgccc vvvdvshedpevkfnwyagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccc vdgvevhnaktkpreeqyaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggnstyrvvsyltylhqdwIng acgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgkayacaysnkalpapiekti tggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcskakgqprepqvytlppsr acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgdeltknqvsltclykgfyps gcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcdiavewesngqpennyktt gagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtappyldsdgsfflyskltydk caccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacsrwqqgnvfscsvmheal ctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagaghnhytqkslslspgk caatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac qvqlvqsgaeykkpgasySEQ ID NO: 73 TSC212 caccggtcaggtccagctggtacagtctggggctgaggtgaagaagcctggkvsckasgytftdyymhw (SEQ ID NO: 74) Scorpionggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactacta vrqapgqglewmgyfnp(huVH#2- catgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatayndytiyaqkfqgrvtmtr VL 107-1A4ttttaatccttataatgattatactagatacgcacagaagttccagggcagagtcdtsistaymelssirsddtav scFv-Fc-accatgaccagggacacgtctatcagcacagcctacatggagctgagcagc yycarsdgyydamdywgCris7 scFv) ctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttactaqgttvtvssggggsggggs cgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcaggtggggsdiqmtqspsamsa ggcggagggtctgggggtggcggatccggaggtggtggctctgatatccasvgdivtitcrasksiskylagatgacccagtctccatccgccatgtctgcatctgtaggagacagagtcaccwfqqkpgkvpklrihsgstlatcacttgccgggcgagtaagagcattagcaaatatttagcctggtttcagcaqsgvpsrfsgsgsgteftltisgaaaccagggaaagttcctaagctccgcatccattctggatctactttgcaatcslqpedfatyycqqhieypaggggtcccatctcggttcagtggcagtggatctgggacagaatttactctcawtfgqgtkveikrassepksccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcatasdkthtcppcpapeaagap ttgaatacccgtggacgttcggccaagggaccaaggtggaaatcaaacgcgsvflfppkpkdtlmisrtpecctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgccc vtcyvvdvshedpevkfnagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccc wyydgvevhnaktkpreeaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggqynstyrvvsyltylhqdwl acgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgngkayacaysnkalpapie tggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcktiskakgqprepqvytlppacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgsrdeltknqvsltclvkgfyp gcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcsdiavewesngqpennyk gagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtattppvldsdgsfflyskltvdcaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac ksrwqqgnvfscsvmheactgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagag lhnhytqkslslspgqrhnncaatgggcagccggagaacaactacaagaccacgcctcccgtgctggact ssintgtqmaghspnsqvqccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtglvqsgggvvqpgrslrlsckgcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaac asgytftrstmhwyrqapgcactacacgcagaagagcctctccctgtctccgggtcagaggcacaacaatt kglewigyinpssaytnyncttccctgaatacaggaactcagatggcaggtcattctccgaattctcaggtccqkfkdrftisadkskstaflq agctggtgcagtctgggggcggagtggtgcagcctgggcggtcactgaggmdslrpedtgvyfcarpqvctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtahydyngfpywgqgtpvtv aggcaggcccctggaaagggtctggaatggattggatacattaatcctagcassggggsggggsggggsa gtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcaqdiqmtqspsslsasvgdr gacaaatccaagagcacagccttcctgcagatggacagcctgaggcccgavtmtcsasssysymnwyq ggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgqkpgkapkrwiydssklas ggtttccttactggggccaagggactcccgtcactgtctctagcggtggcgggyparfsgsgsgtdytltisslagggtctgggggtggcggatccggaggtggtggctctgcacaagacatcc qpalfatyycqqwsrnpptagatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtca fgggtklqitrccatgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagcccggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacac gataa humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac EvqlvqsgaeykkpgatvSEQ ID NO: 75 TSC213 caccggtgaggtccagctggtacagtctggggctgaggtgaagaagcctgkisckasgytftdyymhw (SEQ ID NO: 76) Scorpiongggctacagtgaagatctcctgcaaggcttctggatacacattcactgactact vqqapgkglewmgyfnp(huVH#1- acatgcactgggtgcaacaggcccctggaaaagggcttgagtggatgggatyndytiyaekfqgrvtitadt VL 107-1A4attttaatccttataatgattatactagatacgcagagaagttccagggcagagtstdtaymelsslrsedtavy scFv-Fc-caccataaccgcggacacgtctacagacacagcctacatggagctgagcag ycarsdgyydamdywgqCris7 scFv) cctgagatctgaggacacggccgtgtattactgtgcaagatcggatggttactgttvtvssggggsggggsg acgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcagggggsdiqmtqspsamsas tggcggagggtctgggggtggcggatccggaggtggtggctctgatatccavgdrvtitcrasksiskylawgatgacccagtctccatccgccatgtctgcatctgtaggagacagagtcaccfqqkpgkvpklrihsgstlqatcacttgccgggcgagtaagagcattagcaaatatttagcctggtttcagcasgvpsrfsgsgsgteftltissgaaaccagggaaagttcctaagctccgcatccattctggatctactttgcaatclqpalfatyycqqhieypwaggggtcccatctcggttcagtggcagtggatctgggacagaatttactctcatfgqgtkveikrassepkssccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcatadkthtcppcpapeaagaps ttgaatacccgtggacgttcggccaagggaccaaggtggaaatcaaacgagvflfppkpkdtlmisrtpevtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgccc cvvvdvshedpevkfnwyagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccc vdgvevhnaktkpreeqyaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggnstyrvvsyltvlhqdwing acgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgkayacaysnkalpapiekti tggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcskakgqprepqvytlppsr acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgdeltknqvsltclvkgfyps gcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcdiavewesngqpennyktt gagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtappvldsdgsfflyskltvdk caccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacsrwqqgnvfscsvmheal ctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagaghnhytqkslslspgqrhnns caatgggcagccggagaacaactacaagaccacgcctcccgtgctggactslntgtqmaghspnsqvql ccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtgvqsgggvvqpgrslrlsckagcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaac sgytftrstmhwvrqapgkcactacacgcagaagagcctctccctgtctccgggtcagaggcacaacaatt glewigyinpssaytnynqcttccctgaatacaggaactcagatggcaggtcattctccgaattctcaggtcckfkdrftisadkskstaflqm agctggtgcagtctgggggcggagtggtgcagcctgggcggtcactgaggdslrpedtgvyfcarpqvhyctgtcctgcaaggcttctggctacacctttactagatctacgatgcactgggtadyngfpywgqgtpvtvss aggcaggcccctggaaagggtctggaatggattggatacattaatcctagcaggggsggggsggggsaqd gtgcttatactaattacaatcagaaattcaaggacaggttcacaatcagcgcaiqmtqspsslsasvgdrvt gacaaatccaagagcacagccttcctgcagatggacagcctgaggcccgamtcsasssysymnwyqq ggacaccggcgtctatttctgtgcacggccccaagtccactatgattacaacgkpgkapkrwiydssklasg ggtttccttactggggccaagggactcccgtcactgtctctagcggtggcggvparfsgsgsgtdythisslq agggtctgggggtggcggatccggaggtggtggctctgcacaagacatccpedfatyycqqwsrnpptf agatgacccagtctccaagcagcctgtctgcaagcgtgggggacagggtcagggtklqitr ccatgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagcccggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacac gataa humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac DiqmtqspsamsasvgdrSEQ ID NO: 77 T5C249caccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 78) Scorpiongagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp (huVL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 107-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq 1A4 scFv-acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg Fc-DRA222actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpscFv) ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasykysckasgytftdyy ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcctmhwvrqapgqglewmg ggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgqrhnns gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgslntgtqmaghspnsqvql gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcavesgggvvqpgrslrlsckaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacasgytftrstmhwvrqapgq cgcagaagagcctctccctgtctccgggtcagaggcacaacaattcttccctglewigyinpssaytnynqgaatacaggaactcagatggcaggtcattctccgaattctcaggtccagctggkfkdrftisadkskstaflqmtggagtctgggggcggagtggtgcagcctgggcggtcactgaggctgtcct dslrpedtgvyfcarpqvhygcaaggcttctggctacacctttactagatctacgatgcactgggtaaggcag dyngfpywgqgtpvtvssgcccctggacaaggtctggaatggattggatacattaatcctagcagtgcttatggggsggggsggggsaqd actaattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatciqmtqspsslsasvgdrvt caagagcacagccttcctgcagatggacagcctgaggcccgaggacaccgmtcsasssysymnwyqqgcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttakpgkapkrwiydssklasg ctggggccaagggactcccgtcactgtctctagcggtggcggagggtctggvparfsgsgsgtdythisslq gggtggcggatccggaggtggtggctctgcacaagacatccagatgacccpedfatyycqqwsrnpptf agtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgggtklqitsss gcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacatcctccagct aa humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac DiqmtqspsamsasvgdrSEQ ID NO: 79 T5C250caccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 80) Scorpiongagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp (huVL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 107-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq 1A4 scFv-acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg Fc-DRA222actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpscFv, with ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy H81 linker)ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvrqapgqglewmgggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggtta kthtcppcpapeaagapvctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgevqiplt gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgesyspnsqvqlvesgggvv gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaqpgrslrlsckasgytftrstggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca mhwvrqapgqglewigyicgcagaagagcctctccctgtctccgggtgaagttcaaattcccttgaccgaanpssaytnynqkfkdrftis agttacagcccgaattctcaggtccagctggtggagtctgggggcggagtgadkskstaflqmdslrpedtgtgcagcctgggcggtcactgaggctgtcctgcaaggcttctggctacacct gvyfcarpqvhydyngfpttactagatctacgatgcactgggtaaggcaggcccctggacaaggtctgga ywgqgtpvtvssggggsgatggattggatacattaatcctagcagtgcttatactaattacaatcagaaattcagggsggggsaqdiqmtqs aggacaggttcacaatcagcgcagacaaatccaagagcacagccttcctgcpsslsasvgdrvtmtcsass agatggacagcctgaggcccgaggacaccggcgtctatttctgtgcacggcsysymnwyqqkpgkapk cccaagtccactatgattacaacgggtttccttactggggccaagggactcccrwiydssklasgvparfsgs gtcactgtctctagcggtggcggagggtctgggggtggcggatccggaggtgsgtdytltisslqpedfatyggtggctctgcacaagacatccagatgacccagtctccaagcagcctgtctg ycqqwsrnpptfgggtklqcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaa itsssgttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacatcctccagctaa humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac DiqmtqspsamsasvgdrSEQ ID NO: 81 TSC251caccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 82) Scorpiongagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp (huVL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 107-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq 1A4 scFv-acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg Fc-DRA222actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpscFv, with ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy H83 linker)ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvrqapgqglewmgggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgsslntgt gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgqmaghspnsqvqlvesgg gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagvvqpgrslrlsckasgytftggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca rstmhwvrqapgqglewicgcagaagagcctctccctgtctccgggttcttccctgaatacaggaactcaggyinpssaytnynqkfkdrfatggcaggtcattctccgaattctcaggtccagctggtggagtctgggggcgtisadkskstaflqmdslrpegagtggtgcagcctgggcggtcactgaggctgtcctgcaaggcttctggcta dtgvyfcaipqvhydyngfcacctttactagatctacgatgcactgggtaaggcaggcccctggacaaggt pywgqgtpvtvssggggsctggaatggattggatacattaatcctagcagtgcttatactaattacaatcagaggggsggggsaqdiqmtq aattcaaggacaggttcacaatcagcgcagacaaatccaagagcacagccttspsslsasvgdrvtmtcsascctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgca ssysymnwyqqkpgkapcggccccaagtccactatgattacaacgggtttccttactggggccaagggakrwiydssklasgvparfsg ctcccgtcactgtctctagcggtggcggagggtctgggggtggcggatccgsgsgtdytitisslqpeclfatygaggtggtggctctgcacaagacatccagatgacccagtctccaagcagcc ycqqwsrnpptfgggtklqtgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccagctcaa itsssgtgtaagttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacatcctccagctaa humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac DiqmtqspsamsasvgdrSEQ ID NO: 83 TSC252caccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID NO: 84) Scorpiongagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp (huVL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 107-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq 1A4 scFv-acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg Fc-DRA222actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpscFv, with ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgylftdyy H91 linker)ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvrqapgqglewmgggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndyttyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgnslanq gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgevqipltesyspnsqvqlvegctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcasgggvvqpgrslrlsckasgggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacaytftrstmhwvrqapgqglcgcagaagagcctctccctgtctccgggtaactcattagcaaaccaagaagttewigyinpssaytnynqkfcaaattcccttgaccgaaagttacagcccgaattctcaggtccagctggtggakdrftisadkskstaflqmd gtctgggggcggagtggtgcagcctgggcggtcactgaggctgtcctgcaaslrpedtgvyfcarpqvhydggcttctggctacacctttactagatctacgatgcactgggtaaggcaggccc yngfpywgqgtpvtvssgctggacaaggtctggaatggattggatacattaatcctagcagtgcttatactagggsggggsggggsaqdi attacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaaqmtqspsslsasvgdrvtm gagcacagccttcctgcagatggacagcctgaggcccgaggacaccggcgtcsasssysymnwyqqkptctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactggkapkrwiydssklasgvp gggccaagggactcccgtcactgtctctagcggtggcggagggtctggggarfsgsgsgtdytltisslqpegtggcggatccggaggtggtggctctgcacaagacatccagatgacccagt dfatyycqqwsrnpptfggctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgca gtklqitsssgtgccagctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacatcctccagctaa Humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 157 TSC295caccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID Scorpiongagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp NO: 158) (huVL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 107-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq 1A4 scFv-acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg Fc-DRA222actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpscFv, with ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy H9 linker)ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvrqapgqglewmgggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndyttyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspggsppsp gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgnsqvqlvesgggvvqpgrs gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcalrlsckasgylftrstmhwyrggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacaqapgqglewigyinpssay cgcagaagagcctctccctgtctccgggtgggagcccaccttcaccgaattctnynqkfkdrftisadkskst tcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtaflqmdslipedtgvyfcarcactgaggctgtcctgcaaggcttctggctacacctttactagatctacgatgc pqvhydyngfpywgqgtactgggtaaggcaggcccctggacaaggtctggaatggattggatacattaa pvtvssggggsggggsggtcctagcagtgcttatactaattacaatcagaaattcaaggacaggttcacaatggsaqdiqmtqspsslsas cagcgcagacaaatccaagagcacagccttcctgcagatggacagcctgavgdrvtmtcsasssysymn ggcccgaggacaccggcgtctatttctgtgcacggccccaagtccactatgawyqqkpgkapkrwiydss ttacaacgggtttccttactggggccaagggactcccgtcactgtctctagcgklasgvparfsgsgsgtdytl gtggcggagggtctgggggtggcggatccggaggtggtggctctgcacaatisslqpedfatyycqqwsr gacatccagatgacccagtctccaagcagcctgtctgcaagcgtgggggacnpptfgggtklqitsss agggtcaccatgacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctaca aattacatcctccagctaaHumanized atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatacdiqmtqspsamsasvgdr SEQ ID NO: 159 T5C296caccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID Scorpiongagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp NO: 160) (huVL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 107-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq 1A4 scFv-acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg Fc-DRA222actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpscFv, with ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy H94 linker)ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvrqapgqglewmgggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgsggggs gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgggggsggggspnsqvqlv gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaesgggvvqpgrslrlsckasggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacagytftrstmhwvrqapgqg cgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcglewigyinpssaytnynqk gaggtggctccggcggtggcggatcgccgaattctcaggtccagctggtggfkdrftisadkskstaflqmdagtctgggggcggagtggtgcagcctgggcggtcactgaggctgtcctgca slrpedtgvyfcarpqvhydaggcttctggctacacctttactagatctacgatgcactgggtaaggcaggcc yngfpywgqgtpvtvssgcctggacaaggtctggaatggattggatacattaatcctagcagtgcttatactgggsggggsggggsaqdi aattacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaqmtqspsslsasvgdrvtm agagcacagccttcctgcagatggacagcctgaggcccgaggacaccggctcsasssysymnwyqqkpgtctatttctgtgcacggccccaagtccactatgattacaacgggtttccttactgkapkrwiydssklasgvp ggggccaagggactcccgtcactgtctctagcggtggcggagggtctgggarfsgsgsgtdytltisslqpeggtggcggatccggaggtggtggctctgcacaagacatccagatgaccca dfatyycqqwsrnpptfgggtctccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctg gtklqitssscagtgccagctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacatcctccagcta a Humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac DiqmtqspsamsasvgdrSEQ ID NO: 161 TSC301caccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID Scorpiongagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp NO: 162) (huVL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 107-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq 1A4 scFv-acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg Fc-DRA222actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpscFv, with ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy H105 linker)ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvrqapgqglewmgggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgsggggs gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgggggsggggsqvqlvesg gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggvvqpgrslrlsckasgytggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca ftrstmhwvrqapgqglecgcagaagagcctctccctgtctccgggttctggtggaggcggttcaggcg wigyinpssaytnynqkfkgaggtggctccggcggtggcggatcgcaggtccagctggtggagtctggg drftisadkskstaflqmdslggcggagtggtgcagcctgggcggtcactgaggctgtcctgcaaggcttct rpedtgvyfcarpqvhydyggctacacctttactagatctacgatgcactgggtaaggcaggcccctggac ngfpywgqgtpvtvssggaaggtctggaatggattggatacattaatcctagcagtgcttatactaattacaaggsggggsggggsaqdiq tcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcacmtqspsslsasvgdrvtmt agccttcctgcagatggacagcctgaggcccgaggacaccggcgtctatttccsasssysymnwyqqkp tgtgcacggccccaagtccactatgattacaacgggtttccttactggggccagkapkrwiydssklasgvp agggactcccgtcactgtctctagcggtggcggagggtctgggggtggcgarfsgsgsgtdytltisslqpegatccggaggtggtggctctgcacaagacatccagatgacccagtctccaa dfatyycqqwsrnpptfgggcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgcca gtklqitsssgctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacatcctccagctaa Humanizedatggaagcaccagcgcagcttctcttcctcctgctactctggctcccagatac diqmtqspsamsasvgdrSEQ ID NO: 163 T5C302caccggtgatatccagatgacccagtctccatccgccatgtctgcatctgtagvtitcrasksiskylawfqqk (SEQ ID Scorpiongagacagagtcaccatcacttgccgggcgagtaagagcattagcaaatatttpgkvpklrihsgstlqsgvp NO: 164) (huVL-agcctggtttcagcagaaaccagggaaagttcctaagctccgcatccattctgsrfsgsgsgteftltisslqpe VH#2 107-gatctactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggdfatyycqqhieypwtfgq 1A4 scFv-acagaatttactctcaccatcagcagcctgcagcctgaagattttgcaacttattgtkveikrggggsggggsg Fc-DRA222actgtcaacagcatattgaatacccgtggacgttcggccaagggaccaaggt gggsqvqlvqsgaevkkpscFv, with ggaaatcaaacgaggtggcggagggtctgggggtggcggatccggaggtgasvkvsckasgytftdyy H106 linker)ggtggctctcaggtccagctggtacagtctggggctgaggtgaagaagcct mhwvrqapgqglewmgggggcttcagtgaaggtctcctgcaaggcttctggatacacattcactgactayfnpyndytiyaqkfqgry ctacatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggtmtrdtsistaymelsslrsdatattttaatccttataatgattatactagatacgcacagaagttccagggcagadtavyycarsdgyydamd gtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagcywgqgttvtvsssepkssdagcctgagatctgacgacacggccgtgtattactgtgcaagatcggatggttakthtcppcpapeaagapsv ctacgatgctatggactactggggtcaaggaaccacagtcaccgtctcctcgflfppkpkdtlmisrtpevtcagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac vvvdvshedpevkfnwyctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga vdgvevhnaktkpreeqycaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgnstyrvvsyltvlhqdwing agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagkayacaysnkalpapiekti gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaskakgqprepqvytlppsr ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagdeltknqvsltclvkgfyps gcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaadiavewesngqpennyktt accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctppvldsdgsfflyskltvdk gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctsrwqqgnvfscsvmheal ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgghnhytqkslslspgqrhnns gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacgslntgtqmaghsqvqlves gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggvvqpgrslrlsckasgyggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacatftrstmhwvrqapgqgle cgcagaagagcctctccctgtctccgggtcagaggcacaacaattcttccctwigyinpssaytnynqkfk gaatacaggaactcagatggcaggtcattctcaggtccagctggtggagtctdrftisadkskstaflqmdsl gggggcggagtggtgcagcctgggcggtcactgaggctgtcctgcaaggcrpedtgvyfcarpqvhydyttctggctacacctttactagatctacgatgcactgggtaaggcaggcccctg ngfpywgqgtpvtvssgggacaaggtctggaatggattggatacattaatcctagcagtgcttatactaattaggsggggsggggsaqdiq caatcagaaattcaaggacaggttcacaatcagcgcagacaaatccaagagmtqspsslsasvgdrvtmt cacagccttcctgcagatggacagcctgaggcccgaggacaccggcgtctacsasssysymnwyqqkp tttctgtgcacggccccaagtccactatgattacaacgggtttccttactgggggkapkrwiydssklasgvp ccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtggarfsgsgsgtdytltisslqpecggatccggaggtggtggctctgcacaagacatccagatgacccagtctcc dfatyycqqwsrnpptfggaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgc gtklqitssscagctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatggatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaattacatcctccagctaa

Example 2: Heterodimeric Molecules

PSMA-specific Interceptor molecules were made using Interceptorscaffolding as generally disclosed in International Appl. Nos.PCT/US2010/62436 and PCT/US2010/62404. Briefly, PSMA-specificpolypeptide heterodimers were made by co-expressing two differentpolypeptides chains, one polypeptide chain comprising an immunoglobulinCH1 heterodimerization domain and the other polypeptide chain comprisingan immunoglobulin CL heterodimerization domain. The day beforetransfection HEK293 cells were suspended at a cell concentration of0.5×10⁶ cells/ml in GIBCO® FreeStyle™ 293 expression medium(Invitrogen). 250 mls of cells were used for a large transfection, and60 mls of cells were used for a small transfection. On transfection day,320 ul of 293Fectin™ transfectin reagent (Invitrogen) was mixed with 8mls of media. At the same time, 250 ug of DNA of each of the singlechain polypeptide was mixed with the 8 mls of media and incubated for 5minutes. After 15 minutes of incubation, the DNA-293fectin mixture wasadded to the 250 mls of 293 cells and returned to the shaker at 37° C.and shaken at a speed of 120 RPM. For the smaller transfection using 60mls of cells, a fourth of the DNA, 293fectin, and media were used.

Protein A affinity chromatography was used to purify the proteins. 2 mlof packed protein A agarose (Repligen) was added to a Econo-Column®chromatography column, size 2.5×10 cm (Bio-Rad Laboratories), washedextensively with PBS (10× column volume), and the supernatants wereloaded, washed with PBS again, and eluted with 3 column volumes ofPierce IgG elution buffer. Proteins were then dialyzed extensivelyagainst PBS. Proteins were then concentrated using Amicon® Centricon®centrifugal filter devices (Millipore Corp.) to a final volume around0.5 ml.

Purified proteins were analyzed on a 10% SDS-PAGE gel using XCellSureLock™ Mini-Cell electrophoresis system (Invitrogen).

Bivalent polypeptide heterodimer TSC122 was made by co-expressing singlechain polypeptides TSC084 and TSC093. Single chain polypeptide TSC084comprises from its amino- to carboxyl-terminus: murine 107-1A4(anti-PSMA) VL-VH scFv, human IgG1 SCC-P hinge, human IgG1 CH2, humanIgG1 CH3, and human CH1. The nucleotide and amino acid sequences forTSC084 are set forth in SEQ ID NOs:44 and 46, respectively. Single chainpolypeptide TSC093 comprises from its amino- to carboxyl-terminus: Cris7(anti-CD3) scFv, human IgG1 SCC-P hinge, human IgG1CH2, human IgG1 CH3,and human Cκ(YAE)(i.e., human Cκ without the first Arg or last Cys, butwith N30Y, V55A, and T70E substitutions). The nucleotide and amino acidsequences for TSC093 are set forth in SEQ ID NOs:45 and 47,respectively.

Bivalent polypeptide heterodimer TSC200 was made by co-expressingpolypeptide chains TSC192 and TSC125. TSC192 comprises from its amino-to carboxyl-terminus: humanized 107-1A4 (anti-PSMA) VL-VH#2 scFv, humanIgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3, and human Cκ(YAE). Thenucleotide and amino acid sequences for TSC192 are set forth in SEQ IDNOs:53 and 58, respectively. TSC125 comprises from its amino- tocarboxyl-terminus: Cris7 (anti-CD3) scFv, human IgG1 SCC-P hinge, humanIgG1 CH2, human IgG1 CH3, and human CH1. The nucleotide and amino acidsequences for TSC125 are set forth in SEQ ID NOs:52 and 57,respectively.

Bivalent polypeptide heterodimer TSC202 was made by co-expressingpolypeptide chains TSC193 and TSC125. TSC193 comprises from its amino-to carboxyl-terminus: humanized 107-1A4 (anti-PSMA) VL-VH#1 scFv, humanIgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3, and human Cκ(YAE). Thenucleotide and amino acid sequences for TSC193 are set forth in SEQ IDNOs: 54 and 59, respectively. TSC125 comprises from its amino- tocarboxyl-terminus: Cris7 (anti-CD3) scFv, human IgG1 SCC-P hinge, humanIgG1 CH2, human IgG1 CH3, and human CH1. The nucleotide and amino acidsequences for TSC125 are set forth in SEQ ID NOs:52 and 57,respectively.

Bivalent polypeptide heterodimer TSC204 was made by co-expressingpolypeptide chains TSC195 and TSC093. TSC195 comprises from its amino-to carboxyl-terminus: humanized 107-1A4 (anti-PSMA) VL-VH#2 scFv, humanIgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3, and human CH1. Thenucleotide and amino acid sequences for TSC195 are set forth in SEQ IDNOs:55 and 60, respectively. TSC093 comprises from its amino- tocarboxyl-terminus: Cris7 (anti-CD3) scFv, human IgG1 SCC-P hinge, humanIgG1 CH2, human IgG1 CH3, and human Cκ(YAE). The nucleotide and aminoacid sequences for TSC093 are set forth in SEQ ID NOs: 45 and 47,respectively.

Bivalent polypeptide heterodimer TSC205 was made by co-expressingpolypeptide chains TSC196 and TSC093. TSC196 comprises from its amino-to carboxyl-terminus: humanized 107-1A4 (anti-PSMA) VL-VH#1 scFv, humanIgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3, and human CH1. Thenucleotide and amino acid sequences for TSC196 are set forth in SEQ IDNOs:56 and 61, respectively. TSC093 comprises from its amino- tocarboxyl-terminus: Cris7 (anti-CD3) scFv, human IgG1 SCC-P hinge, humanIgG1 CH2, human IgG1 CH3, and human Cκ(YAE). The nucleotide and aminoacid sequences for TSC093 are set forth in SEQ ID NOs: 45 and 47,respectively.

Example 3: SCORPION Molecule Construction

PSMA-specific SCORPION molecules (TSC194 (SEQ ID NO:48 (nucleic acid),SEQ ID NO:49 (amino acid); TSC199 (SEQ ID NO:50 (nucleic acid), SEQ IDNO:51 (amino acid)); TSC 212 (SEQ ID NO:73 (nucleic acid), SEQ ID NO:74(amino acid)); TSC213 (SEQ ID NO:75 (nucleic acid), SEQ ID NO:76 (aminoacid)); TSC249 (SEQ ID NO:77 (nucleic acid), SEQ ID NO:78 (amino acid));TSC250 (SEQ ID NO:79 (nucleic acid), SEQ ID NO:80 (amino acid)); TSC251(SEQ ID NO:81 (nucleic acid), SEQ ID NO:82 (amino acid)); and TSC252(SEQ ID NO:83 (nucleic acid), SEQ ID NO:84 (amino acid))) were madeusing standard molecular biology techniques, starting with existingSCORPION scaffolding as templates and using the methods generallydisclosed in, e.g., PCT Application Publication No. WO 2007/146968, U.S.Patent Application Publication No. 2006/0051844, PCT ApplicationPublication No. WO 2010/040105, PCT Application Publication No. WO2010/003108, and U.S. Pat. No. 7,166,707 (see also Table 3). Insertionof the N-terminal scFv binding domain was accomplished through digestionof the parental template and scFv insert with either the restrictionenzymes HindIII and XhoI or AgeI and XhoI, desired fragments wereidentified and isolated by agarose gel purification, and ligation.Insertion of the C-terminal scFv binding domain was accomplished throughdigestion of the parental template and scFv insert with the restrictionenzymes EcoRI and NotI, desired fragments were identified and isolatedby agarose gel purification, and ligation.

Example 4: Binding of Chimeric and Humanized Molecules to PSMA(+) andPSMA(−) Cell Lines

Monoclonal antibodies were purified from hybridoma cell culture media bystandard procedures. SMIP, Interceptor, and SCORPION molecules disclosedherein were produced by transient transfection of human HEK293 cells,and purified from cell culture supernatants by Protein A affinitychromatography. If aggregates were detected after affinitychromatography, secondary size exclusion chromatography was alsoperformed to ensure homogeneity of the protein.

Binding studies on PSMA+ (C4-2, Wu et al., 1994 Int. J. Cancer57:406-12) and PSMA− (DU-145, Stone et al., 1978, Intl. J. Cancer21:274-81) prostate cancer cell lines were performed by standardFACS-based staining procedures. A typical experiment would label 300,000cells per well with a range of 200 nM to 0.1 nM binding molecule in 100ul of FACS buffer (PBS+2% normal goat serum+2% fetal bovine serum+0.1%sodium azide) on ice, followed by washes and incubation withfluorescently-labeled secondary antibody, goat anti-human IgG (1:400dilution of Invitrogen #11013=5 ug/ml). After washing secondary antibodyoff cells, cells were incubated with 7-Aminoactinomycin D (7-AAD)staining solution (BD Pharmingen™ cat#559925)(6 ul of 7AAD to 100 ul ofFACS Buffer) for 20 minutes. Signal from bound molecules was detectedusing a FACSCalibur™ flow cytometer (BD Biosciences) and analyzed byFlowJo flow cytometry analysis software. 7-AAD+ cells were excluded fromanalysis. Nonlinear regression analysis to determine EC50s was performedin GraphPad Prism® graphing and statistics software.

Binding studies (FIG. 1) were used to compare the parent 107-1A4 murineantibody (i.e., TSC045) with the chimeric SMIP molecules (TSC085,TSC092) and the bispecific, chimeric Interceptor molecule TSC122. Bothchimeric SMIP molecules showed comparable affinity to PSMA+ cells as theparent murine antibody, although one (TSC085, with a VL-VH scFvorientation) showed a lower level of saturation on the surface of thecell. The bispecific, chimeric Interceptor molecule (TSC122), which hadonly a single 107-1A4 binding domain, showed a lower binding affinity tothe PSMA+ cells. All showed little to no binding to the PSMA− cell lineDU-145. Binding studies of humanized SMIP molecules TSC188 and TSC189(FIG. 2A) showed comparable affinities to those previously determined(data not shown) for the parent monoclonal antibody, with similarly highlevels of saturation and selectivity for PSMA+ C4-2 cells over PSMA−DU-145 cells. Binding studies of humanized SCORPION molecules TSC194 andTSC199 showed comparable affinities to the parental humanized SMIPmolecules TSC188 and TSC189 (FIG. 2B), with no binding to the PSMA−DU-145 cell line.

Example 5: Differential Cellular Internalization Seen Between 107-1A4Antibody, SMIP and Interceptor Scaffolds

The binding proteins for internalization studies were directly labeledwith CypHer™5E Mono NHS Ester (GE Healthcare, #PA15401) according tomanufacturer's instructions. CypHer5E is a pH-sensitive red excited dyethat fluoresces at low pH, which is typically encountered inside ofendosomes and lysosomes; CypHer5E fluorescence can be used as a proxyfor cellular internalization as a result. Dye dissolved in fresh DMSOwas added to purified protein in PBS/sodium carbonate buffer, pH 8.3(9:1), at a dye:protein molar ratio of 20:1. After at least 1 hourincubation in the dark at room temperature, labeled protein wasseparated from unreacted dye by dialysis at 4° C. Absorbance at 280 nmand 500 nm was used to calculate protein and dye concentration for thelabeled material. The resulting dye:protein ratio ranged from 6 to 14,and this value was used to normalize the imaging data. To ensure thatthe presence of protein aggregates did not bias the internalizationdata, when individual molecules had detectable levels of aggregates(>5%), secondary size exclusion chromatography was used to purifymolecules to very high levels of homogeneity (>95%).

Cells were plated 2 days before experiment at 4000 cells per well inpoly-D-lysine-coated 96-well plates, black with clear bottoms (BDBiocoat, 356640) in usual culture media. Media changes during experimentwere conducted carefully to maintain cell adhesion to surface. Nucleiwere stained with Hoechst 33342 (Invitrogen, H3570) in serum-free phenolred-free RPMI media (Invitrogen, 11835) plus 20 mM HEPES (Invitrogen,15630) (called PRF-RPMI) for an hour. Wells were washed with PRF-RPMIplus 10% FBS, and 100 ul warm PRF-RPMI+10% FBS was added. Plates weremoved to ice for 5 minutes then labeled binding proteins at variousdilutions were added from 5× stock solutions for one hour binding onice. Plates were moved to a 37° C. CO₂ incubator for 60 minutes to allowinternalization to proceed. Before imaging, media was replaced withPRF-RPMI+1% FBS.

Wells were scanned on IN Cell Analyzer 1000 automated cellular andsubcellular imaging system (GE Healthcare) to quantitate internalizedprotein, data was collected from 8 fields in each well. The acquisitionprotocol was set to collect data with suitable filter sets for Hoechstand CypHer5E, and bright field images. Data was analyzed by IN CellInvestigator software, using a protocol developed to detect fluorescentgranules within a zone of cytoplasm encircling each nucleus andmeasuring their area. Total granule area detected was normalized tocompensate for the relative level of dye substitution per labeledprotein.

Internalization experiments using the parental 107-1A4 murine antibody,or the chimeric SMIP and Interceptor molecules, showed nointernalization in the PSMA− DU-145 cell line (data not shown), but someinternalization could be detected on the PSMA+ LNCaP (CRL-1740™,American Type Culture Collection) or C4-2 cell lines (FIG. 3).Internalization of the parental antibody was greater than the SMIP orInterceptor molecules at all concentrations tested. Apparentinternalization from the (monovalent) Interceptor molecule was higherthan from the (bivalent) SMIP molecule, which could be due to the higherpotential binding stoichiometry—each Interceptor molecule can onlyengage one molecule of PSMA, whereas each SMIP molecule can engage twomolecules of PSMA, potentially leading to twice as much Interceptormolecule accumulating on the cell surface. If both the Interceptor andSMIP molecules have similar levels of internalization, a higher apparentsignal would always be seen from the Interceptor molecule.

Example 6: Redirected T-Cell Cytotoxicity Against PSMA(+) Cell Lines

Peripheral blood mononuclear cells (PBMC) were isolated from human bloodfrom two different donors (labeled as AG or VV) using standard ficollgradients. The isolated cells were washed in saline buffer. T cells wereadditionally isolated using a Pan T-cell Isolation Kit II from MiltenyiBiotec (Bergisch Gladbach, Germany) using the manufacturer's protocol. Tcells were used with or without stimulation, as noted in the figures(see FIGS. 4-6), and added at a 10:1 ratio (T cell:target cell) unlessindicated otherwise.

C4-2 castration-resistant prostate cancer (CRPC) cells were labeled withCellTracker™ Green cytoplasmic dye (Invitrogen, C7025) followingmanufacturer's protocol in order to distinguish them from T cells.Labeled C4-2 cells were seeded into poly-D-lysine-coated 96-well plates,as used in Example 3, at 8000 cells per well in standard growth media,one day before addition of T cells and Interceptor molecule. Ten ul ofconcentrated bispecific Interceptor molecule (TSC122, TSC200, TSC202, orTSC204) was added to 100 ul of media per well, plus 50 ul of T cells(80,000 cells) in standard growth media. Cell cultures were kept in CO₂incubator at 37° C. overnight. After 24 hr exposure to Interceptormolecule, cells were stained with 7-AAD and Hoechst dyes to enablequantitation of dead cells. Media was changed to 100 ul RPMI+1% FBS+10ug/ml 7-AAD+Hoechst at 1:1000 dilution of stock, and incubated for anadditional 30 minutes.

Imaging and quantitation was performed by use of an InCell Analyzermicroscope (GE), collecting data from 10 fields per well. Theacquisition protocol was set to collect data with suitable filter setsfor: a) nuclei detection via Hoechst stain, b) cell type discriminationvia CellTracker™ Green detection, c) live/dead cell status determinationvia 7-AAD staining, and bright field images. Quantitation was performedby InCell Workstation software, using a decision tree application.Individual cells were detected by presence of nuclear stain by Hoechst.Threshold values of signal in the green channel (CellTracker™ Green)were used to split cells into C4-2 (positive) and T cell (negative)populations. Threshold values of signal in red channel (7-AAD) were usedto split cells into dead (positive) and live (negative) populations.

Bispecific Interceptor molecules featuring either the 107-1A4 murinescFv or humanized 107-1A4 scFv as well as an anti-CD3 scFv (Cris7) weretested for the ability to cross-link T-cells to target PSMA+ tumor cellsand enable target-dependent cytotoxic T cell responses (so-called‘redirected T cell cytotoxicity’, or RTCC). Potent target-dependentcytotoxic activity over 24 hours was observed with the chimeric TSC122Interceptor molecule (FIG. 4) with T-cells from two different donors;roughly 60% of target cells were lysed by treatment with as little as100 pM of TSC122 Interceptor molecule. No direct cytotoxicity on PSMA+cells was observed in the absence of effector T-cells (FIG. 4); nocytotoxicity was similarly observed on PSMA− cells in the presence ofeffector T-cells (data not shown). The cytotoxic activity of humanizedInterceptor molecules (TSC200, TSC202, and TSC204) was also testedalongside the parent chimeric Interceptor molecule (TSC122) (FIG. 5).Two of the humanized Interceptor molecules (TSC200, TSC204) showed lowerRTCC activity than the parent; one humanized Interceptor molecule(TSC202) showed equal RTCC activity to the parent chimeric Interceptormolecule (TSC122) with similar, low pM potency. These resultsdemonstrated that certain humanized Interceptor molecules had similar Tcell cytotoxicity as the parental chimeric Interceptor molecule.

The cytotoxic activity of humanized SCORPION molecules (TSC194, TSC199,TSC212, TSC213), compared to that of the chimeric Interceptor moleculeTSC122, was also examined (FIG. 6). The humanized SCORPION moleculeswith anti-PSMA scFvs in the VL-VH orientation (TSC194, TSC199) hadcomparable cytotoxic activity to the bispecific chimeric Interceptormolecule (also with an scFv in the VL-VH orientation, and also includingan anti-CD3 scFv (Cris7)). The humanized SCORPION molecules withanti-PSMA scFvs in the VH-VL orientation, on the other hand, had loweroverall cytotoxicity.

Example 7: Target-Dependent T-Cell Activation and Proliferation InducedAgainst PSMA+ Cell Lines Directed by Bispecific 107-1A4-DerivedMolecules

To compare the effectiveness of different bispecific polypeptidemolecules at inducing target-dependent T-cell activation andproliferation, four different anti-PSMA and anti-CD3 bispecificmolecules including TSC122 (a chimeric Interceptor molecule), TSC202(humanized Interceptor molecule), TSC194 (a humanized SCORPIONmolecule), and TSC199 (a humanized SCORPION molecule) were compared.

C4-2 prostate cancer cells (PSMA+) were obtained from MD Anderson CancerCenter (Houston, Tex.) and cultured according to the provided protocol.Peripheral blood mononuclear cells (PBMC) were isolated from human bloodusing standard ficoll gradients. The isolated cells were washed insaline buffer. T cells were further isolated using a Pan T-cellIsolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) usingthe manufacturer's protocol.

Proliferation was assessed by labeling isolated T cell populations withcarboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE-labeled Tcells were plated in U-bottom 96-well plates at 100,000 cells/well,respectively, with 30,000 C4-2 tumor cells/well, to achieve T cell totumor cell ratios of roughly 3:1. Concentrations of test moleculesranging from 10 nM to 0.1 pM were added to the cell mixtures in a totalof 200 ul/well in RPMI 1640 media supplemented with 10% human or bovineserum, sodium pyruvate and non-essential amino acids. Plates wereincubated at 37° C., 5% CO₂ in humidified incubators. After 3 days,cells were labeled with antibodies for flow cytometric analysis. Cellswere labeled and washed in their original plates to minimize cell lossesduring transfers, and all labeling was done in saline buffer with 0.2%bovine serum albumin. First, cells were pre-incubated with 100 ug/mlhuman IgG at room temperature for 15 min. Subsequently, cells wereincubated with a mixture (total volume 50 ul) of the followingdye-labeled antibodies: CD5-PE, CD4-APC, CD8-Pacific Blue, CD25-PE-Cy7,as well as 7-Amino Actinomycin D (7AAD hereafter) for 40 min. Cells werewashed twice, resuspended in 80 to 120 ul volumes, and measuredimmediately in a BD LSRII flow cytometer to acquire 80% of the contentsof each well. The sample files were analyzed using FlowJo software tocalculate the percentages and numbers of cells that had undergone atleast one cell division, according to their CFSE profile, by gatingsequentially on activated, live CD4+ or CD8+ T cells (7AAD−, CD5+ CD25+CD4+ or 7AAD− CD5+ CD25+ CD8+, respectively). Mean values and standarddeviations were calculated using Microsoft Excel software. Graphs wereplotted using Microsoft Excel or GraphPad Prism.

Analysis of live CD4+ and CD8+ populations from wells with C4-2 cellstreated with T-cells (FIG. 7A and FIG. 7B) revealed a significantincrease in both the total number of cells and percent proliferatingcells in the presence of C4-2 cells displaying the target PSMA antigen.Proliferation was slightly higher for CD4+ T-cells than CD8+ T-cells,and the proliferation induced by the Interceptor molecules TSC122 andTSC202 saturated at a higher level than the responses induced by theSCORPION molecules TSC194 and TSC199. All molecules showed induction ofT-cell proliferation at low concentrations (100 pM). No significantdifferences in relative induction of CD4+ versus CD8+ cell proliferationwere apparent between molecules.

Example 8: Competitive Binding Studies of Anti-PSMA Molecules Confirms107-1A4 Binds a Unique Epitope on PSMA

To show that anti-PSMA murine monoclonal antibody 107-1A4, chimeric107-1A4 SMIP molecule (TSC085) and humanized 107-1A4 SMIP molecule(TSC189) binds a unique epitope on PSMA, which is not recognized bycommon literature antibodies (J415, J591), and that the conversion ofmurine monoclonal antibody 107-1A4 to SMIP format did not result in ashift in that binding epitope, competition binding experiments werecarried out. Hybridomas producing the J591, Hu591 (a humanized versionof J591) and J415 antibodies were obtained from ATCC. Monoclonalantibodies were purified from hybridoma cell culture media by standardprocedures. SMIP molecules were produced by transient transfection ofhuman 293 cells, and purified from cell culture supernatants by ProteinA affinity chromatography. If aggregates were detected after affinitychromatography, secondary size exclusion chromatography was alsoperformed to ensure homogeneity of the protein.

Competitive binding studies on the PSMA+ C4-2 prostate cancer cell linewere performed by standard FACS-based staining procedures. To simplifybinding measurements, molecules with human Fc domains were used tocompete against molecules with murine Fc domains, and either ananti-human or anti-mouse antibody was used to detect binding to thetarget cell line.

In a typical experiment, molecule X (binder) would be mixed withmolecule Y (competitor), placed on ice, and then used to label 300,000cells per well with 4 nM of molecule X and a range of 250 nM to 0.4 nMmolecule Y in 100 ul of FACS buffer (PBS+2% normal goat serum+2% fetalbovine serum+0.1% sodium azide) on ice, followed by washes andincubation with fluorescently-labeled secondary antibody specific formolecule X, either goat anti-human IgG (1:400 dilution of Invitrogen11013=5 ug/ml) or goat anti-mouse IgG (1:400 dilution of Invitrogen11017). After washing secondary antibody off cells, cells were incubatedwith 7AAD (6 ul of BD Pharmingen 7AAD, cat#559925) to 100 ul of FACSBuffer) for 20 minutes. Signal from bound molecules was detected using aFACSCalibur flow cytometer and analyzed by FlowJo. 7AAD+ cells wereexcluded from analysis. Nonlinear regression analysis to determine EC50swas performed in GraphPad Prism.

Competitive binding studies (FIG. 8A) were used to see if the humanizedJ591 antibody, Hu591, could compete with the binding of 107-1A4, J591 orJ415 murine antibodies to cells. No competition was observed for thebinding of 107-1A4, suggesting it binds a non-competitive epitope;competition was observed for the binding of both J591 and J415antibodies, however. Next, additional binding studies were carried outto see if the three murine antibodies could compete with the binding ofthe chimeric 107-1A4 SMIP molecule TSC085 to cells (FIG. 8B). Strongcompetition was seen from binding of the parental 107-1A4 antibody,confirming that the SMIP molecule bound to the same epitope. Noeffective competition was seen from the J591 or J415 murine antibodies.Last, binding studies were carried out to see if the three murineantibodies could compete with the binding of the humanized 107-1A4 SMIPmolecule TSC189 to cells (FIG. 8C). Again, similarly strong competitionwas seen from binding of the parental 107-1A4 antibody, but no effectivecompetition was seen from the J591 or J415 murine antibodies. Thisconfirms that 107-1A4 binds a unique epitope on PSMA. It also shows thatany shift in behavior of 107-1A4-based SMIP and Interceptor moleculesfrom that of the parental 107-1A4 antibody is not due to a shift inbinding epitopes.

Example 9: Inhibition of Tumor Growth In Vivo Using an Anti-PSMABispecific Molecule

To confirm the effectiveness of an anti-PSMA bispecific molecule of thepresent disclosure (e.g., anti-PSMA and anti-CD3 bispecific molecules)at inhibiting tumor growth in vivo, the anti-PSMA bispecific molecule isevaluated as follows.

Prophylactic Treatment, or Prevention of Tumor Engraftment ofSubcutaneous Tumors:

Cultured, PSMA-expressing tumor cell lines (such as LNCaP, LNCaP C4-2,LNCaP C4-2B, VCaP, CWR22Rv1, LAPC4, MDA-PCa-2b, LuCaP 23.1, LuCaP 58,LuCaP 70, LuCaP 77) are mixed with human lymphocytes (either humanperipheral blood mononuclear cells or purified T-cells) and injectedsubcutaneously into immunodeficient mice (such as SCID, NOD/SCID, etc).An anti-PSMA bispecific molecule is injected intravenously on the day ofinjection and on several subsequent days. Dose-dependent inhibition oftumor outgrowth, as assessed by tumor volume, indicates that therespective molecule has efficacy against PSMA-expressing tumors in vivo.

Therapeutic Treatment, or Regression of Previously EstablishedSubcutaneous Tumors:

Cultured, PSMA-expressing tumor cell lines (such as LNCaP, LNCaP C4-2,LNCaP C4-2B, VCaP, CWR22Rv1, LAPC4, MDA-PCa-2b, LuCaP 23.1AI, LuCaP 58,LuCaP 70, LuCaP 77) are injected subcutaneously into immunodeficientmice (such as SCID, NOD/SCID, etc). Tumor growth is monitored, and thestudy is initiated when tumors show signs of established growth(typically a volume of ˜200 mm3). Human lymphocytes (either humanperipheral blood mononuclear cells or purified T-cells) are injectedintravenously along with an anti-PSMA bispecific molecule on the day ofinjection. The anti-PSMA bispecific molecule is injected severalsubsequent days. Dose-dependent inhibition of tumor growth, as assessedby tumor volume, indicates that the respective molecule has efficacyagainst PSMA-expressing tumors in vivo.

Prophylactic Treatment, or Prevention of Tumor Engraftment ofIntra-Tibial Tumors:

Cultured, PSMA-expressing tumor cell lines (such as LNCaP C4-2, LNCaPC4-2B, VCaP, CWR22Rv1, LAPC4, MDA-PCa-2b, LuCaP 23.1, LuCaP 58, LuCaP70, LuCaP 77) are mixed with human lymphocytes (either human peripheralblood mononuclear cells or purified T-cells) and injected intra-tibiallyinto immunodeficient mice (such as SCID, NOD/SCID, etc). An anti-PSMAbispecific molecule is injected intravenously on the day of injectionand on several subsequent days. Dose-dependent inhibition of tumorgrowth, as assessed by serum biomarkers, radiography, fluorescentimaging, weight loss, and other proxy measurements of tumor volume,indicates that the respective molecule has efficacy againstPSMA-expressing tumors in vivo.

Therapeutic Treatment, or Regression of Previously EstablishedIntra-Tibial Tumors:

Cultured, PSMA-expressing tumor cell lines (such as LNCaP C4-2, LNCaPC4-2B, VCaP, CWR22Rv1, LAPC4, MDA-PCa-2b, LuCaP 23.1AI, LuCaP 58, LuCaP70, LuCaP 77) are injected intra-tibially into immunodeficient mice(such as SCID, NOD/SCID, etc). Tumor growth is monitored, and the studyis initiated when tumors show signs of established growth (typically avolume of ˜200 mm3). Human lymphocytes (either human peripheral bloodmononuclear cells or purified T-cells) are injected intravenously alongwith an anti-PSMA bispecific molecule on the day of injection. Theanti-PSMA bispecific molecule is injected several subsequent days.Dose-dependent inhibition of tumor growth, as assessed by serumbiomarkers, radiography, fluorescent imaging, weight loss, and otherproxy measurements of tumor volume, indicates that the respectivemolecule has efficacy against PSMA expressing tumors in vivo.

1.-153. (canceled)
 154. A prostate-specific membrane antigen(PSMA)-binding polypeptide comprising a humanized PSMA-binding domain, afirst hinge region, an immunoglobulin constant region and a secondbinding domain, wherein the PSMA binding domain comprises: (i) animmunoglobulin light chain variable region comprising LCDR1, LCDR2, andLCDR3, and (ii) an immunoglobulin heavy chain variable region comprisingHCDR1, HCDR2, and HCDR3, wherein the LCDR1, LCDR2 and LCDR3 have theamino acid sequences set forth in SEQ ID NOs: 15, 16 and 17,respectively, and the HCDR1, HCDR2, and HCDR3 have the amino acidsequences set forth in SEQ ID NOs: 9, 10 and 11, respectively; whereinthe second binding domain specifically binds a T cell receptor (TCR)complex or a component thereof; and wherein the PSMA-binding polypeptideis capable of inducing redirected T cell cytotoxicity (RTCC) against acell expressing PSMA.
 155. The PSMA-binding polypeptide of claim 154,wherein the second binding domain specifically binds CD3.
 156. ThePSMA-binding polypeptide of claim 154, wherein the immunoglobulinconstant region comprises immunoglobulin CH2 and CH3 domains of IgG1,IgG2, IgG3, IgG4, IgA1, IgA2 or IgD.
 157. The PSMA-binding polypeptideof claim 154, wherein the PSMA-binding domain comprises an amino acidsequence that is at least 95% identical to the amino acid sequence setforth in SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:70, or SEQ ID NO:72.
 158. The PSMA-binding polypeptide of claim 154,wherein the PSMA-binding polypeptide comprises, in order fromamino-terminus to carboxyl-terminus or in order from carboxyl-terminusto amino-terminus, (a) the PSMA binding domain, (b) the first hingeregion, (c) the immunoglobulin constant region, (d) a second hingeregion, and (e) the second binding domain.
 159. The PSMA-bindingpolypeptide of claim 158, wherein the first hinge region and/or thesecond hinge region is derived from (i) a stalk region of a type II Clectin or (ii) an immunoglobulin hinge region.
 160. The PSMA-bindingpolypeptide of claim 154, wherein the light and heavy chain variableregions of the second binding domain are selected from the groupconsisting of: (a) a light chain variable region comprising an aminoacid sequence that is at least 95% identical to the amino acid sequenceset forth in residues 139-245 of SEQ ID NO:47 and comprisingcomplementarity determining regions (CDRs) comprising the amino acidsequences of the corresponding CDRs set forth in residues 139-245 of SEQID NO:47 and a heavy chain variable region comprising an amino acidsequence that is at least 95% identical to the amino acid sequence setforth in residues 1-121 of SEQ ID NO:47 and comprising CDRs comprisingthe amino acid sequences of the corresponding CDRs set forth in residues1-121 of SEQ ID NO:47; (b) a light chain variable region comprising anamino acid sequence that is at least 95% identical to the amino acidsequence set forth in residues 634-740 of SEQ ID NO:78 and comprisingCDRs comprising the amino acid sequences of the corresponding CDRs setforth in residues 634-740 of SEQ ID NO:78 and a heavy chain variableregion comprising an amino acid sequence that is at least 95% identicalto the amino acid sequence set forth in residues 496-616 of SEQ ID NO:78and comprising CDRs comprising the amino acid sequences of thecorresponding CDRs set forth in residues 496-616 of SEQ ID NO:78; and(c) a light chain variable region comprising an amino acid sequence thatis at least 95% identical to the amino acid sequence set forth in SEQ IDNO:86 and comprising CDRs comprising the amino acid sequences of thecorresponding CDRs set forth in SEQ ID NO:86 and a heavy chain variableregion comprising an amino acid sequence that is at least 95% identicalto the amino acid sequence set forth in SEQ ID NO:87 and comprising CDRscomprising the amino acid sequences of the corresponding CDRs set forthin SEQ ID NO:87.
 161. The PSMA-binding polypeptide of claim 154, whereinsaid PSMA-binding polypeptide comprises an amino acid sequence that isat least 95% identical to the amino acid sequence set forth in SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, or SEQ ID NO:164.
 162. The PSMA-binding polypeptide of claim154, wherein the PSMA-binding domain is a single chain Fv (scFv). 163.The PSMA-binding polypeptide of claim 162, wherein the light chainvariable region and heavy chain variable region of the scFv are joinedby an amino acid sequence comprising (Gly₄Ser)_(n), wherein n=1-5 (SEQID NO:165).
 164. The PSMA-binding polypeptide of claim 162, wherein thescFv comprises an amino acid sequence that is at least 95% identical tothe amino acid sequence set forth in SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:34, or SEQ ID NO:35.
 165. A dimericPSMA-binding protein comprising first and second polypeptide chains,wherein each of said polypeptide chains is the PSMA-binding polypeptideof claim
 154. 166. A composition comprising the PSMA-binding protein ofclaim 165 and a pharmaceutically acceptable carrier, diluent, orexcipient.
 167. An isolated nucleic acid encoding the PSMA-bindingpolypeptide of claim
 154. 168. A recombinant host cell comprising thenucleic acid of claim
 167. 169. A PSMA-binding polypeptide comprising ahumanized PSMA-binding domain, a first hinge region, an immunoglobulinconstant region and a second binding domain, wherein the PSMA bindingdomain comprises: (i) an immunoglobulin light chain variable regioncomprising LCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavychain variable region comprising HCDR1, HCDR2, and HCDR3, wherein theLCDR1, LCDR2 and LCDR3 have the amino acid sequences set forth in SEQ IDNOs: 15, 16 and 17, respectively, and the HCDR1, HCDR2, and HCDR3 havethe amino acid sequences set forth in SEQ ID NOs: 9, 10 and 11,respectively; wherein the second binding domain specifically binds a Tcell receptor (TCR) complex or a component thereof; wherein theimmunoglobulin light chain variable region comprises an amino acidsequence that is at least 95% identical to the amino acid sequence setforth in SEQ ID NO:5 or SEQ ID NO:23 and the heavy chain variable regioncomprises an amino acid sequence that is at least 95% identical to theamino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:25, or SEQ IDNO:27; and wherein the PSMA-binding polypeptide is capable of inducingRTCC against a cell expressing PSMA.
 170. The PSMA-binding polypeptideof claim 169, wherein the light chain variable region comprises theamino acid sequence set forth in SEQ ID NO:23 and the heavy chainvariable region comprises the amino acid sequence set forth in SEQ IDNO:25 or SEQ ID NO:27.
 171. A method for treating a disorder in asubject, wherein the disorder is characterized by overexpression of PSMAand wherein the disorder is a cancer, a prostate disorder, or aneovascular disorder, the method comprising administering to the subjecta therapeutically effective amount of the dimeric PSMA-binding proteinof claim 165 under conditions whereby RTCC in the subject is induced.172. The method of claim 171, wherein the disorder is prostate cancer,colorectal cancer, gastric cancer, castrate-resistant prostate cancer,benign prostatic hyperplasia, solid tumor growth, clear cell renalcarcinoma, colorectal cancer, bladder cancer, or lung cancer.